Bifunctional molecules for degradation of egfr and methods of use

ABSTRACT

The present application provides bifunctional compounds of Formula (I):which act as protein degradation inducing moieties for EGFR and/or a mutant thereof. The present application also describes methods for the targeted degradation of EGFR and/or a mutant thereof through the use of the bifunctional compounds that link a ubiquitin ligase-binding moiety to a ligand that is capable of binding to EGFR and/or a mutant thereof which can be utilized in the treatment of disorders modulated by EGFR or a mutant thereof.

STATEMENT OF RELATED INVENTIONS

This application is a continuation of U.S. patent application Ser. No.16/588,750, filed Sep. 30, 2019, which is a continuation of U.S. patentapplication Ser. No. 16/163,254, filed Oct. 17, 2018, which is acontinuation of International Patent Application No. PCT/US2017/028950,filed with the Patent Cooperation Treaty, U.S. Receiving Office, on Apr.21, 2017, which claims the benefit of U.S. Provisional PatentApplication No. 62/326,574, filed Apr. 22, 2016. The entirety of thisapplication is hereby incorporated by reference for all purposes.

GOVERNMENT SUPPORT

This invention was made with government support under grant number P01CA154303 awarded by The National Institutes of Health. The governmenthas certain rights in the invention.

BACKGROUND OF THE DISCLOSURE

Ubiquitin-Proteasome Pathway (UPP) is a critical pathway that regulatesproteins and degrades misfolded or abnormal proteins. UPP is central tomultiple cellular processes, and if defective or imbalanced, leads topathogenesis of a variety of diseases. The covalent attachment ofubiquitin to specific protein substrates is achieved through the actionof E3 ubiquitin ligases. These ligases comprise over 500 differentproteins and are categorized into multiple classes defined by thestructural element of their E3 functional activity. For example,cereblon (CRBN) interacts with damaged DNA binding protein 1 and formsan E3 ubiquitin ligase complex with Cullin 4 in which the proteinsrecognized by CRBN are ubiquitinated and degraded by proteasomes.Various immunomodulatory drugs (IMiDs), e.g. thalidomide andlenalidomide, binds to CRBN and modulate CRBN's role in theubiquitination and degradation of protein factors involved inmaintaining regular cellular function.

Harnessing the ubiquitin-proteasome pathway for therapeutic interventionhas received significant interest from the scientific community. Thepublication by Gosink et al. (Proc. Natl. Acad. Sci. USA 1995, 92,9117-9121) titled “Redirecting the Specificity of Ubiquitination byModifying Ubiquitin-Conjugating Enzymes” showed proof of concept invitro that engineered peptides can selectively direct ubiquitination tointracellular proteins. The publication by Nawaz et al. (Proc. Natl.Acad. Sci. U.S.A. 1999, 96, 1858-1862) titled “Proteasome-DependentDegradation of the Human Estrogen Receptor” describes ER degradation asa target for the ubiquitin-proteasome pathway. The publication by Zhouet al. (Mol. Cell 2000, 6, 751-756) titled “Harnessing theUbiquitination Machinery to Target the Degradation of Specific CellularProteins” demonstrated an engineered receptor capable of directingubiquitination in mammalian and yeast cells.

U.S. Pat. No. 6,306,663 filed in 1999 assigned to Proteinex, Inc.,titled “Controlling Protein Levels in Eucaryotic Organisms” appears tobe the first patent disclosure of ubiquitinating molecules thatincorporate a ubiquitination recognition element and a target proteinrecognition element.

U.S. Pat. No. 7,041,298 titled “Proteolysis Targeting ChimericPharmaceutical” was filed in September 2000 by Deshales et al. andgranted in May 2006. The publication by Sakamoto et al. (Proc. Natl.Acad. Sci. USA 2001, 98, 8554-8559) titled “Protacs: Chimeric MoleculesThat Target Proteins to the Skp1-Cullin-F Box Complex for Ubiquitinationand Degradation” describes a “PROTAC” consisting of a small moleculebinder of MAP-AP-2 linked to a peptide capable of binding the F-boxprotein β-TRCP, the disclosure of which is also provided in thecorresponding U.S. Pat. No. 7,041,298. The publication by Sakamoto etal. (Mol. Cell. Proteomics 2003, 2, 1350-1358) titled “Development ofProtacs to Target Cancer-Promoting Proteins for Ubiquitination andDegradation” describes an analogous PROTAC (PROTAC2) that instead ofdegrading MAP-AP-2, degrades estrogen and androgen receptors. Thepublication by Schneekloth et al. (J. Am. Chem. Soc. 2004, 126,3748-3754) titled “Chemical Genetic Control of Protein Levels: Selectivein Vivo Targeted Degradation” describes an analogous degradation agent(PROTAC3) that targets the FK506 binding protein (FKBP12) and shows thatboth PROTAC2 and PROTAC3 hit their respective targets using greenfluorescent protein (GFP) imaging. The publication by Schneekloth et al.(ChemBioChem 2005, 6, 40-46) titled “Chemical Approaches to ControllingIntracellular Protein Degradation” described the state of the field atthe time. The publication by Schneekloth et al. (Bioorg. Med. Chem.Lett. 2008, 18, 5904-5908) titled “Targeted Intracellular ProteinDegradation Induced by a Small Molecule: En Route to ChemicalProteomics” describes a degradation agent that consists of two smallmolecules linked by PEG that in vivo degrades the androgen receptor byconcurrently binding the androgen receptor and Ubiquitin E3 ligase. WO2013/170147 filed by Crews et al. titled “Compounds Useful for PromotingProtein Degradation and Methods Using Same” describes compoundscomprising a protein degradation moiety covalently bound to a linker,wherein the C log P of the compound is equal to or higher than 1.5. Areview by Buckley et al. (Angew. Chem. Int. Ed. Engl. 2014, 53,2312-2330) titled “Small-Molecule Control of Intracellular ProteinLevels through Modulation of the Ubiquitin Proteasome System” describesa variety of publications. WO 2015/160845 assigned to Arvinas Inc.titled “Imide Based Modulators of Proteolysis and Associated methods ofUse” describes the use of degradation compounds including thalidomide toutilize cereblon as the E3 ligase protein. The publication by Lu et al.(Chem. Biol. 2015, 22, 755-763) titled “Hijacking the E3 UbiquitinLigase Cereblon to Efficiently Target Brd4” describes thalidomide baseddegradation compounds useful for degrading BRD4. Additional publicationsinclude Bondeson et al. (Nat. Chem. Biol. 2015, 11, 611-617) titled“Catalytic in Vivo Protein Knockdown by Small-Molecule Protacs”;Gustafson et al. (Angewandte Chemie, International Edition in English2015, 54, 9659-9662) titled “Small-Molecule-Mediated Degradation of theAndrogen Receptor through Hydrophobic Tagging”; Buckley et al. (J. Am.Chem. Soc. 2012, 134, 4465-4468) titled “Targeting the Von Hippel-LindauE3 Ubiquitin Ligase Using Small Molecules to Disrupt the Vhl/Hif-1alphaInteraction”; U.S. 2016/0058872 assigned to Arvinas Inc. titled “ImideBased Modulators of Proteolysis and Associated Methods of Use”; U.S.2016/0045607 assigned to Arvinas Inc. titled “Estrogen-related ReceptorAlpha Based PROTAC Compounds and Associated Methods of Use”; U.S.2014/0356322 assigned to Yale University, GlaxoSmithKline, and CambridgeEnterprise Limited University of Cambridge titled “Compounds and Methodsfor the Enhanced Degradation of Targeted Proteins & Other Polypeptidesby an E3 Ubiquitin Ligase”; Lai et al. (Angewandte Chemie, InternationalEdition in English 2016, 55, 807-810) titled “Modular Protac Design forthe Degradation of Oncogenic Bcr-Abl”; and Toure et al. (Angew. Chem.Int. Ed. 2016, 55, 1966-1973) titled “Small-Molecule Protacs: NewApproaches to Protein Degradation”. See also US 2016/0176916, US2016/0235730, US 2016/0235731, US 2016/0243247, WO 2016/105518, WO2016/077380, WO2016/105518, WO 2016/077375, WO2017/007612, andWO2017/024317.

It was discovered and reported in 2010 that thalidomide binds tocereblon (see Ito et al. (Science 2010, 327, 1345-1350) titled“Identification of a Primary Target of Thalidomide Teratogenicity” andFischer et al. (Nature 2014, 512, 49-53) titled “Structure of theDdb1-Crbn E3 Ubiquitin Ligase in Complex with Thalidomide”). Itoh et al.also described a small molecule linked to a peptide that utilizes E3ubiquitin ligase to degrade retinoic acid-binding proteins. (See J. Am.Chem. Soc. 2010, 132, 5820-5826 titled “Protein Knockdown Using MethylBestatin-Ligand Hybrid Molecules: Design and Synthesis of Inducers ofUbiquitination-Mediated Degradation of Cellular Retinoic Acid-BindingProteins”).

The object of the present invention is to provide bifunctional compoundsand compositions for the treatment of serious diseases, including kinasemediated disorders.

SUMMARY

The present application provides novel bifunctional compounds, whichfunction to recruit targeted proteins to E3 ubiquitin ligase fordegradation, and methods of preparation and uses thereof. Thebifunctional compound is of Formula X:

wherein:

the Targeting Ligand is capable of binding to a targeted protein, suchas EGFR and/or a mutant EGFR;

the Linker is a group that covalently binds to the Targeting Ligand andthe Degron; and

the Degron is capable of binding to a ubiquitin ligase, such as an E3ubiquitin ligase.

In one embodiment, the E3 ubiquitin ligase is cereblon.

The present application also provides targeted degradation of proteinsthrough the use of bifunctional compounds, including bifunctionalcompounds that link an E3 ubiquitin ligase-binding moiety to a ligandthat binds the targeted proteins.

The present application also describes a bifunctional compound ofFormula Y:

or an enantiomer, diastereomer, stereoisomer, or pharmaceuticallyacceptable salt thereof optionally in a pharmaceutically acceptablecarrier,wherein:

A is phenyl or pyridinyl;

X is CH, C(C₁-C₃) alkyl, or N;

R¹ is H or (C₁-C₃) alkyl;

R² is (C₆-C₁₀) aryl, or heteroaryl comprising one or two 5- to7-membered rings and 1-4 heteroatoms selected from N, O, and S, whereinthe aryl and heteroaryl are each optionally substituted with one or moreR⁴;

each R⁴ is independently selected from (C₁-C₄) alkyl, (C₁-C₄) haloalkyl,(C₁-C₄) alkoxy, (C₁-C₄) haloalkoxy, halogen, NO₂, OH, CN, C(O)R⁶,C(O)OR⁶, C(O)NR⁶R⁷, NR⁶R⁷, (C₃-C₇) cycloalkyl, heterocyclyl comprising a5- to 7-membered ring and 1-3 heteroatoms selected from N, O, and S,(C₆-C₁₀) aryl, and heteroaryl comprising one or two 5- to 7-memberedrings and 1-4 heteroatoms selected from N, O, and S, wherein the alkyl,cycloalkyl, heterocyclyl, aryl, and heteroaryl are each optionallysubstituted with one or more R⁵;

each R⁵ is independently selected from (C₁-C₄) alkyl, (C₁-C₄) haloalkyl,(C₁-C₄) alkoxy, (C₁-C₄) haloalkoxy, halogen, NO₂, OH, and CN;

each R⁶ is independently H or (C₁-C₃) alkyl;

each R⁷ is independently H or (C₁-C₃) alkyl;

R³ is (C₁-C₃) alkyl or

X² is N or CR⁸;

R⁸ is H, (C₁-C₄) alkyl, (C₁-C₄) haloalkyl, (C₁-C₄) alkoxy, (C₁-C₄)haloalkoxy, halogen, NO₂, NH₂, OH, or CN;

each R⁹ is independently selected from (C₁-C₄) alkyl, (C₁-C₄) haloalkyl,(C₁-C₄) alkoxy, (C₁-C₄) haloalkoxy, halogen, NO₂, NH₂, OH, and CN;

n is 0 or 1; and

p is 0, 1, 2, or 3;

the Linker is a group that covalently binds to

and the Degron;

the Degron is capable of binding to a ubiquitin ligase; and

the Targeting Ligand is capable of binding to EGFR or a mutant EGFR.

In one embodiment, the E3 ubiquitin ligase is cereblon.

In one embodiment, the Targeting Ligand is capable of binding to EGFR.

In one embodiment, the Targeting Ligand is capable of binding to amutant EGFR.

In a further embodiment, the Targeting Ligand is capable of binding to aT790M/L858R EGFR mutant.

In a further embodiment, the Targeting Ligand is capable of binding to aT790M/L858R/C797S EGFR mutant.

The present application describes novel bifunctional compounds, whichfunction to recruit targeted proteins to E3 ubiquitin ligase fordegradation, and methods of preparation and uses thereof.

In one embodiment the bifunctional compound is of Formula Z:

or an enantiomer, diastereomer, stereoisomer, or pharmaceuticallyacceptable salt thereof optionally in a pharmaceutically acceptablecarrier,wherein:

the Targeting Ligand is selected from

the Linker is a group that covalently binds to the Targeting Ligand andthe Degron; and

the Degron is capable of binding to a ubiquitin ligase.

In one embodiment, the E3 ubiquitin ligase is cereblon.

In one embodiment the bifunctional compound is of Formula A:

or an enantiomer, diastereomer, stereoisomer, or pharmaceuticallyacceptable salt thereof optionally in a pharmaceutically acceptablecarrierwherein:

the Targeting Ligand is selected from

the Linker is a group that covalently binds to the Targeting Ligand andthe Degron; and

the Degron is capable of binding to a ubiquitin ligase.

In one embodiment, the E3 ubiquitin ligase is cereblon.

In one embodiment the bifunctional compound is of Formula B:

or an enantiomer, diastereomer, stereoisomer, or pharmaceuticallyacceptable salt thereof optionally in a pharmaceutically acceptablecarrierwherein:

the Targeting Ligand-Linker is selected from

and

the Degron is capable of binding to a ubiquitin ligase.

In one embodiment, the E3 ubiquitin ligase is cereblon.

In one embodiment the bifunctional compound is of Formula C:

or an enantiomer, diastereomer, stereoisomer, or pharmaceuticallyacceptable salt thereof optionally in a pharmaceutically acceptablecarrierwherein:

the Targeting Ligand-Linker is selected from

and

the Degron is capable of binding to a ubiquitin ligase.

In one embodiment, the E3 ubiquitin ligase is cereblon.

The present application also describes targeted degradation of proteinsthrough the use of bifunctional compounds, including bifunctionalcompounds that link an E3 ubiquitin ligase-binding moiety to a ligandthat binds the targeted proteins.

The present application also provides a bifunctional compounds ofFormula D.

Formula D is a compound selected from

or an enantiomer, diastereomer, stereoisomer, or pharmaceuticallyacceptable salt thereof optionally in a pharmaceutically acceptablecarrier.

The present application further includes a Degron of Formula D1:

or an enantiomer, diastereomer, or stereoisomer thereof, wherein Y, Z¹,R¹³, R¹⁴, R¹⁵, R¹⁶, v, and q are each as defined herein.

The present application further includes a Linker of Formula L0:

or an enantiomer, diastereomer, or stereoisomer thereof, wherein p1, p2,p3, W, Q, and Z are each as defined herein, the Linker is covalentlybonded to a Degron with the

next to Q, and covalently bonded to a Targeting Ligand with the

next to Z.

The present application also describes a pharmaceutical compositioncomprising a therapeutically effective amount of a bifunctional compoundof Formula X, Formula Y, Formula Z, Formula A, Formula B, Formula C, orFormula D or an enantiomer, diastereomer, stereoisomer, orpharmaceutically acceptable salt thereof, and a pharmaceuticallyacceptable carrier.

The present application provides a method of modulating or decreasingthe amount of a kinase, comprising administering to a subject in needthereof a therapeutically effective amount of a bifunctional compound ofFormula X, Formula Y, Formula Z, Formula A, Formula B, Formula C, orFormula D, or a pharmaceutically acceptable salt, hydrate, solvate,prodrug, stereoisomer, or tautomer thereof, or a pharmaceuticalcomposition of the application.

The present application provides a method of modulating or decreasingthe amount of epidermal growth factor receptor (EGFR) and/or a mutantthereof, comprising administering to a subject in need thereof atherapeutically effective amount of a bifunctional compound of FormulaX, Formula Y, Formula Z, Formula A, Formula B, Formula C, or Formula D,or a pharmaceutically acceptable salt, hydrate, solvate, prodrug,stereoisomer, or tautomer thereof, or a pharmaceutical composition ofthe application.

The present application provides a method of treating or preventing adisease in which EGFR or a mutant thereof plays a role or a diseaseresistant to an EGFR targeted therapy, such as a therapy with gefitinib,erlotinib, AZD9291, CO-1686 or WZ4002, comprising administering to asubject in need thereof a therapeutically effective amount of abifunctional compound of Formula X, Formula Y, Formula Z, Formula A,Formula B, Formula C, or Formula D, or a pharmaceutically acceptablesalt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, or apharmaceutical composition of the application.

The present application provides a method of treating or preventingcancer, comprising administering to a subject in need thereof atherapeutically effective amount of a bifunctional compound of FormulaX, Formula Y, Formula Z, Formula A, Formula B, Formula C, or Formula D,or a pharmaceutically acceptable salt, hydrate, solvate, prodrug,stereoisomer, or tautomer thereof, or a pharmaceutical composition ofthe application.

The present application provides a method of treating or preventingcancer in a subject in need thereof, wherein the cancer cell comprisesan activated EGFR or a mutant thereof or wherein the subject isidentified as being in need of inhibition of EGFR or a mutant thereoffor the treatment or prevention of cancer, comprising administering tothe subject a therapeutically effective amount of a bifunctionalcompound of Formula X, Formula Y, Formula Z, Formula A, Formula B,Formula C, or Formula D, or a pharmaceutically acceptable salt, hydrate,solvate, prodrug, stereoisomer, or tautomer thereof, or a pharmaceuticalcomposition of the application.

The present application provides a method of treating or preventingcancer in a subject in need thereof, wherein the cancer cell comprisesan activated ERBB2 or wherein the subject is identified as being in needof inhibition of ERBB2 for the treatment or prevention of cancer,comprising administering to a subject in need thereof a therapeuticallyeffective amount of a bifunctional compound of Formula X, Formula Y,Formula Z, Formula A, Formula B, Formula C, or Formula D, or apharmaceutically acceptable salt, hydrate, solvate, prodrug,stereoisomer, or tautomer thereof, or a pharmaceutical composition ofthe application.

The present application provides a kit comprising a compound capable ofmodulating or decreasing the amount of EGFR or a mutant thereof,selected from a bifunctional compound of Formula X, Formula Y, FormulaZ, Formula A, Formula B, Formula C, or Formula D, or a pharmaceuticallyacceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomerthereof.

The present application provides a bifunctional compound of Formula X,Formula Y, Formula Z, Formula A, Formula B, Formula C, or Formula D, ora pharmaceutically acceptable salt, hydrate, solvate, prodrug,stereoisomer, or tautomer thereof, or a pharmaceutical composition ofthe application for use in modulating or decreasing the amount of akinase.

The present application provides a bifunctional compound of Formula X,Formula Y, Formula Z, Formula A, Formula B, Formula C, or Formula D, ora pharmaceutically acceptable salt, hydrate, solvate, prodrug,stereoisomer, or tautomer thereof, or a pharmaceutical composition ofthe application for use in modulating or decreasing the amount of EGFRand/or a mutant thereof.

The present application provides a bifunctional compound of Formula X,Formula Y, Formula Z, Formula A, Formula B, Formula C, or Formula D, ora pharmaceutically acceptable salt, hydrate, solvate, prodrug,stereoisomer, or tautomer thereof, or a pharmaceutical composition ofthe application for use in treating or preventing a disease in whichEGFR or a mutant thereof plays a role, or a disease resistant to an EGFRtargeted therapy, such as a therapy with gefitinib, erlotinib, AZD9291,CO-1686 or WZ4002.

The present application provides a bifunctional compound of Formula X,Formula Y, Formula Z, Formula A, Formula B, Formula C, or Formula D, ora pharmaceutically acceptable salt, hydrate, solvate, prodrug,stereoisomer, or tautomer thereof, or a pharmaceutical composition ofthe application for use in treating or preventing cancer.

The present application relates a bifunctional compound of Formula X,Formula Y, Formula Z, Formula A, Formula B, Formula C, or Formula D, ora pharmaceutically acceptable salt, hydrate, solvate, prodrug,stereoisomer, or tautomer thereof, or a pharmaceutical composition ofthe application for use in treating or preventing cancer in a subject,wherein the subject is identified as being in need of inhibition of EGFRor a mutant thereof for the treatment or prevention of cancer, orwherein the cancer cell comprises an activated EGFR or a mutant thereof.

The present application provides a bifunctional compound of Formula X,Formula Y, Formula Z, Formula A, Formula B, Formula C, or Formula D, ora pharmaceutically acceptable salt, hydrate, solvate, prodrug,stereoisomer, or tautomer thereof, or a pharmaceutical composition ofthe application for use in treating or preventing cancer in a subject,wherein the subject is identified as being in need of inhibition ofERBB2 for the treatment or prevention of cancer, or wherein the cancercell comprises an activated ERBB2.

The present application provides a bifunctional compound of Formula X,Formula Y, Formula Z, Formula A, Formula B, Formula C, or Formula D, ora pharmaceutically acceptable salt, hydrate, solvate, prodrug,stereoisomer, or tautomer thereof, or a pharmaceutical composition ofthe application for use in the manufacture of a medicament formodulating or decreasing the amount of a kinase.

The present application provides a bifunctional compound of Formula X,Formula Y, Formula Z, Formula A, Formula B, Formula C, or Formula D, ora pharmaceutically acceptable salt, hydrate, solvate, prodrug,stereoisomer, or tautomer thereof, or a pharmaceutical composition ofthe application for use in the manufacture of a medicament formodulating or decreasing the amount of EGFR and/or a mutant thereof.

The present application provides a bifunctional compound of Formula X,Formula Y, Formula Z, Formula A, Formula B, Formula C, or Formula D, ora pharmaceutically acceptable salt, hydrate, solvate, prodrug,stereoisomer, or tautomer thereof, or a pharmaceutical composition ofthe application for use in the manufacture of a medicament for treatingor preventing a disease in which EGFR or a mutant thereof plays a role,or a disease resistant to an EGFR targeted therapy, such as a therapywith gefitinib, erlotinib, AZD9291, CO-1686 or WZ4002.

The present application provides a bifunctional compound of Formula X,Formula Y, Formula Z, Formula A, Formula B, Formula C, or Formula D, ora pharmaceutically acceptable salt, hydrate, solvate, prodrug,stereoisomer, or tautomer thereof, or a pharmaceutical composition ofthe application for use in the manufacture of a medicament for treatingor preventing cancer.

The present application provides a bifunctional compound of Formula X,Formula Y, Formula Z, Formula A, Formula B, Formula C, or Formula D, ora pharmaceutically acceptable salt, hydrate, solvate, prodrug,stereoisomer, or tautomer thereof, or a pharmaceutical composition ofthe application for use in the manufacture of a medicament for treatingor preventing cancer in a subject, wherein the subject is identified asbeing in need of inhibition of EGFR or a mutant thereof for thetreatment or prevention of cancer, or wherein the cancer cell comprisesan activated EGFR or a mutant thereof.

The present application provides a bifunctional compound of Formula X,Formula Y, Formula Z, Formula A, Formula B, Formula C, or Formula D, ora pharmaceutically acceptable salt, hydrate, solvate, prodrug,stereoisomer, or tautomer thereof, or a pharmaceutical composition ofthe application for use in the manufacture of a medicament for treatingor preventing cancer in a subject, wherein the subject is identified asbeing in need of inhibition of ERBB2 for the treatment or prevention ofcancer, or wherein the cancer cell comprises an activated ERBB2.

The present application further provides compounds and compositions withan improved efficacy and/or safety profile relative to known EGFRinhibitors. The present application also provides agents with novelmechanisms of action toward EGFR kinases in the treatment of varioustypes of diseases including cancer and metastasis.

The details of the disclosure are set forth in the accompanyingdescription below. Although methods and materials similar or equivalentto those described herein can be used in the practice or testing of thepresent application, illustrative methods and materials are nowdescribed. Other features, objects, and advantages of the disclosurewill be apparent from the description and from the claims. Unlessotherwise defined, all technical and scientific terms used herein areaccorded the meaning commonly known to one with ordinary skill in theart. In the specification and the appended claims, the singular formsalso include the plural unless the context clearly dictates otherwise.Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. All patents and publicationscited in this specification are incorporated herein by reference intheir entireties.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing EGFR activity in BaF3 cells expressing theEGFR/L858R mutant after the cells were treated with variousconcentrations of TL compound 1 (1), TL compound 2 (2), Compound I-1(3), or Compound I-3 (4). The x-axis is concentration measured in M andthe y-axis is EGFR activity measured as a percent of the control.

FIG. 2 is a graph showing EGFR activity in BaF3 cells expressing theEGFR L858R/T790M mutant after the cells were treated with variousconcentrations of TL compound 1, Compound I-1, or Compound I-2. Thex-axis is concentration measured in M and the y-axis is EGFR activitymeasured as a percent of the control.

FIG. 3 is a western blot showing the levels of EGFR and tubulin in BaF3cells expressing the EGFR L858R/T790M mutant after an 8-hour treatmentwith various concentrations of Compound I-1, in the absence or presenceof cetuximab (1 μg/ml).

FIG. 4 is a graph showing EGFR activity in BaF3 cells expressing theEGFR L858R mutant after the cells were treated with variousconcentrations of EAI045 (an inhibitor of L858R/T790M EGFR mutants), TLcompound 1, TL compound 2, Compound I-1, or Compound I-3. The x-axis isconcentration measured in M and the y-axis is EGFR activity measured asa percent of the control.

FIG. 5A is a western blot quantifying the levels of phosphorylated EGFR(pEGFR), EGFR, and tubulin in BaF3 cells expressing the EGFR L858R/T790Mmutant over the course of 72 hours when exposed to Compound I-1 andCompound I-18.

FIG. 5B is a western blot quantifying the levels of phosphorylated EGFR(pEGFR), EGFR, and tubulin in BaF3 cells expressing the EGFR L858R/T790Mmutant after an 8-hour treatment of increasing concentrations ofCompound I-1 and Compound I-18.

FIG. 6 is a western blot quantifying levels of phosphorylated EGFR(pEGFR), EGFR, phosphorylated AKt, Akt, phosphorylated ERK1, ERK2,phosphorylated ERK1, phosphorylated ERK2, and tubulin in apatient-derived lung cancer cell line (H1975) harboring the EGFRL858R/T790M mutant after a 24-hour treatment of increasingconcentrations of intermediate 2-48A, Compound I-32, Compound I-22, andCompound I-18. The compounds were compared to known EGFR inhibitorAZD9291 tested at a concentration of 1 μM.

FIG. 7 is a western blot quantifying the levels of EGFR protein andtubulin after a 4 hour pretreatment of either DMSO, thalidomide (10 μM),or TL compound 1 (10 μM) followed by 24 hour Compound I-1 treatment inthe presence or absence of 1 μg/mL cetuximab in BaF3 cells expressingthe EGFR L858R/T790M.

FIG. 8 is a western blot quantifying levels of phosphorylated EGFR(pEGFR), EGFR, and tubulin in BaF3 cells expressing the EGFR L858R/T790Mmutant after a 4-hour pretreatment of the NEDD8-Activating Enzyme (NAE)inhibitor MLN4924 at various concentrations in the presence or absenceof a 24-hour treatment of Compound I-1.

DETAILED DESCRIPTION EGFR Target Protein

The epidermal growth factor receptor (EGFR, Erb-B1) belongs to a familyof proteins, involved in the proliferation of normal and malignantcells. Overexpression of Epidermal Growth Factor Receptor (EGFR) ispresent in at least 70% of human cancers. Epidermal growth factorreceptor (EGFR) tyrosine kinase inhibitors (TKIs) are effectivetherapies for EGFR mutant advanced non-small cell lung cancer (NSCLC)patients. However, the vast majority of patients will develop diseaseprogression following successful treatment with an EGFR TKI. Onemechanism of acquired resistance is a substitution of leucine forarginine at position 858 (L858R) that occurs within exon 21 of thereceptor. The most common mechanism of acquired resistance, detected in60% of patients, is a secondary mutation in EGFR at position T790(T790M). This mutation leads to an increase in ATP affinity and makes itmore difficult for reversible EGFR TKIs (e.g., first-generationinhibitors gefitinib and erlotinib and second-generation inhibitorsafatinib, neratinib, and dacomitinib) to bind the EGFR TKI domain.

All current EGFR TKIs target the ATP site, and while third generationirreversible inhibitors can overcome T790M, they are all renderedimpotent by the C797S mutation, which is already arising in treatedpatients. Third-generation inhibitors include osimertinib (Tagrisso),rociletinib (CO-1686), olmutinib (Olita; BI 1482694; HM61713),naquotinib (ASP8273), nazartinib (EGF816), and PF-06747775. Cetuximab,an anti-EGFR antibody that blocks receptor dimerization, is noteffective in EGFR-mutant NSCLC because mutational activation of thekinase is effectively “downstream” of receptor dimerization.

Recently, a fourth-generation inhibitor knowns as EAI045 has beendiscovered that is selective for drug-resistant EGFR mutants overwild-type EGFR. EAI045 is currently being developed as an inhibitor ofthe C797S mutation (Wang S, et al. Cancer Lett. 2017, 385:51-54).

Additionally, small molecule inhibitors of EGFR have been identifiedincluding thiazole-containing inhibitors as described in WO2017/004383filed by Dana-Farber Cancer Institute. When tested in an EGFRT790M/L858R Ba/F3 cell line, a number of compounds were efficacious ininducing inhibition of the mutant cell line and potent compounds werereported to have an IC₅₀ value of less than 0.5 μM.

The present application describes bifunctional compounds having utilityas modulators of ubiquitination and proteosomal degradation of targetedproteins, especially compounds comprising a moiety capable of binding toa polypeptide or a protein that is degraded and/or otherwise inhibitedby the bifunctional compounds of the present invention. In particular,the present application is directed to compounds that contain asmall-molecule moiety capable of binding to an E3 ubiquitin ligase, suchas cereblon, and a ligand capable of binding to a target protein in sucha way that the target protein is placed in proximity to the ubiquitinligase to effect degradation (and/or inhibition) of that protein. In oneembodiment, the small molecule moiety has a molecular weight below2,000, 1,000, 500, or 200 Daltons. In one embodiment, the small moleculemoiety is a thalidomide-like moiety. In one embodiment, the E3 ubiquitinligase is cereblon.

Compounds of the Application

The present application provides novel bifunctional compounds, whichfunction to recruit targeted proteins to E3 ubiquitin ligase fordegradation, and methods of preparation and uses thereof. Thebifunctional compound is of Formula X:

wherein:

the Targeting Ligand is capable of binding to a targeted protein, suchas EGFR and/or a mutant EGFR;

the Linker is a group that covalently binds to the Targeting Ligand andthe Degron; and

the Degron is capable of binding to a ubiquitin ligase, such as an E3ubiquitin ligase.

In one embodiment, the E3 ubiquitin ligase is cereblon.

The present application also provides targeted degradation of proteinsthrough the use of bifunctional compounds, including bifunctionalcompounds that link an E3 ubiquitin ligase-binding moiety to a ligandthat binds the targeted proteins.

The present application also describes a bifunctional compound ofFormula Y:

or an enantiomer, diastereomer, stereoisomer, or pharmaceuticallyacceptable salt thereof optionally in a pharmaceutically acceptablecarrier,wherein:

R¹, R², R³, A, X, and n are each as defined herein;

the Linker is a group that covalently binds to

and the Degron;

the Degron is capable of binding to a ubiquitin ligase; and

the Targeting Ligand is capable of binding to EGFR or a mutant EGFR.

In one embodiment, the E3 ubiquitin ligase is cereblon.

In one embodiment, the Targeting Ligand is capable of binding to EGFR.

In one embodiment, the Targeting Ligand is capable of binding to amutant EGFR.

In a further embodiment, the Targeting Ligand is capable of binding to aT790M/L858R EGFR mutant.

In a further embodiment, the Targeting Ligand is capable of binding to aT790M/L858R/C797S EGFR mutant.

In one embodiment the bifunctional compound is of Formula Z:

or an enantiomer, diastereomer, stereoisomer, or pharmaceuticallyacceptable salt thereof optionally in a pharmaceutically acceptablecarrier,wherein:

the Targeting Ligand is selected from

the Linker is a group that covalently binds to the Targeting Ligand andthe Degron; and

the Degron is capable of binding to a ubiquitin ligase.

In one embodiment, the E3 ubiquitin ligase is cereblon.

In one embodiment the bifunctional compound is of Formula A:

or an enantiomer, diastereomer, stereoisomer, or pharmaceuticallyacceptable salt thereof optionally in a pharmaceutically acceptablecarrier,

wherein:

the Targeting Ligand is selected from

the Linker is a group that covalently binds to the Targeting Ligand andthe Degron; and

the Degron is capable of binding to a ubiquitin ligase.

In one embodiment, the E3 ubiquitin ligase is cereblon.

In one embodiment the bifunctional compound is of Formula B:

or an enantiomer, diastereomer, stereoisomer, or pharmaceuticallyacceptable salt thereof optionally in a pharmaceutically acceptablecarrier,

wherein:

the Targeting Ligand-Linker is selected from

and

the Degron is capable of binding to a ubiquitin ligase.

In one embodiment, the E3 ubiquitin ligase is cereblon.

In one embodiment the bifunctional compound is of Formula C:

or an enantiomer, diastereomer, stereoisomer, or pharmaceuticallyacceptable salt thereof optionally in a pharmaceutically acceptablecarrier,

wherein:

the Targeting Ligand-Linker is selected from

and

the Degron is capable of binding to a ubiquitin ligase.

In one embodiment, the E3 ubiquitin ligase is cereblon.

The present application also describes targeted degradation of proteinsthrough the use of bifunctional compounds, including bifunctionalcompounds that link an E3 ubiquitin ligase-binding moiety to a ligandthat binds the targeted proteins.

Targeting Ligand

Targeting Ligand (TL) (or target protein moiety or target protein ligandor ligand) is a small molecule which is capable of binding to a targetprotein of interest, such as EGFR or a mutant thereof.

In one embodiment, a Targeting Ligand is a compound of Formula TL-I:

or an enantiomer, diastereomer, stereoisomer, or pharmaceuticallyacceptable salt thereof,wherein:

A is phenyl or pyridinyl;

X is CH, C(C₁-C₃) alkyl, or N;

R¹ is H or (C₁-C₃) alkyl;

R² is (C₆-C₁₀) aryl, or heteroaryl comprising one or two 5- to7-membered rings and 1-4 heteroatoms selected from N, O, and S, whereinthe aryl and heteroaryl are each optionally substituted with one or moreR⁴;

each R⁴ is independently selected from (C₁-C₄) alkyl, (C₁-C₄) haloalkyl,(C₁-C₄) alkoxy, (C₁-C₄) haloalkoxy, halogen, NO₂, OH, CN, C(O)R⁶,C(O)OR⁶, C(O)NR⁶R⁷, NR⁶R⁷, (C₃-C₇) cycloalkyl, heterocyclyl comprising a5- to 7-membered ring and 1-3 heteroatoms selected from N, O, and S,(C₆-C₁₀) aryl, and heteroaryl comprising one or two 5- to 7-memberedrings and 1-4 heteroatoms selected from N, O, and S, wherein the alkyl,cycloalkyl, heterocyclyl, aryl, and heteroaryl are each optionallysubstituted with one or more R⁵;

each R⁵ is independently selected from (C₁-C₄) alkyl, (C₁-C₄) haloalkyl,(C₁-C₄) alkoxy, (C₁-C₄) haloalkoxy, halogen, NO₂, OH, and CN;

each R⁶ is independently H or (C₁-C₃) alkyl;

each R⁷ is independently H or (C₁-C₃) alkyl;

R³ is (C₁-C₃) alkyl or

X² is N or CR⁸;

R⁸ is H, (C₁-C₄) alkyl, (C₁-C₄) haloalkyl, (C₁-C₄) alkoxy, (C₁-C₄)haloalkoxy, halogen, NO₂, NH₂, OH, or CN;

each R⁹ is independently selected from (C₁-C₄) alkyl, (C₁-C₄) haloalkyl,(C₁-C₄) alkoxy, (C₁-C₄) haloalkoxy, halogen, NO₂, NH₂, OH, and CN;

n is 0 or 1; and

p is 0, 1, 2, or 3;

wherein the Targeting Ligand is bonded to a Linker via the

next to

In some embodiments, X is N. In some embodiments, X is CH. In someembodiments, X is C(CH₃), C(CH₂CH₃), or C(CH₂CH₂CH₃).

In some embodiments, A is phenyl. In some embodiments, A is pyridinyl.

In some embodiments, n is 0. In some embodiments, n is 1.

In some embodiments, R³ is (C₁-C₃) alkyl. In some embodiments, R³ isselected from methyl, ethyl, propyl, or i-propyl. In one embodiment, R³is methyl.

In some embodiments, R³ is

(e.g., 2-pyridinyl, 3-pyridinyl, or 4-pyridinyl). In some embodiments,R³ is

In some embodiments, X² is N. In some embodiments, X² is CR⁸.

In some embodiments, R⁸ is H. In some embodiments, R⁸ is (C₁-C₄) alkyl(e.g., methyl, ethyl, propyl, i-propyl, or butyl), (C₁-C₄) haloalkyl(e.g., CH₂F, CHF₂, or CF₃), (C₁-C₄) alkoxy (e.g., methoxy, ethoxy,propoxy, or butoxy), (C₁-C₄) haloalkoxy (e.g., OCH₂F, OCHF₂, or OCF₃),halogen (e.g., F, Cl, Br, or I), NO₂, NH₂, OH, or CN. In someembodiments, R⁸ is H, NO₂, NH₂, OH, or F. In some embodiments, R⁸ is OHor F.

In some embodiments, p is 0. In some embodiments, p is 1. In someembodiments, p is 2. In some embodiments, p is 3. In some embodiments, pis 0, 1, or 2. In some embodiments, p is 0 or 1. In some embodiments, pis 1 or 1. In some embodiments, p is 1, 2, or 3.

In some embodiments, at least one R⁹ is (C₁-C₄) alkyl (e.g., methyl,ethyl, propyl, i-propyl, or butyl), (C₁-C₄) haloalkyl (e.g., CH₂F, CHF₂,or CF₃), (C₁-C₄) alkoxy (e.g., methoxy, ethoxy, propoxy, or butoxy),(C₁-C₄) haloalkoxy (e.g., OCH₂F, OCHF₂, or OCF₃), halogen (e.g., F, Cl,Br or I), NO₂, NH₂, OH, or CN. In some embodiments, at least one R⁹ isNO₂, NH₂, OH, or F. In some embodiments, at least one R⁹ is OH or F.

In some embodiments, R¹ is H. In some embodiments, R¹ is (C₁-C₃) alkyl(e.g., methyl, ethyl, propyl, or i-propyl). In some embodiments, R¹ isH, methyl, or ethyl. In some embodiments, R¹ is H or methyl. In someembodiments, R¹ is H.

In some embodiments, R² is (C₆-C₁₀) aryl optionally substituted with oneor more R⁴. In some embodiments, R² is phenyl optionally substitutedwith one or more R⁴.

In some embodiments, R² is heteroaryl comprising one or two 5- to7-membered rings and 1-4 heteroatoms selected from N, O, and S (e.g.,pyrrolyl, pyrazolyl, imidazolyl, triazolyl, oxazolyl, isoxazolyl,oxadiazolyl, dioxazolyl, thiazolyl, isothiazolyl, thiadiazolyl,dithiazolyl, thiophenyl, pyridinyl, pyridazinyl, pyrimidinyl, triazinyl,benzothiazolyl, benzoimidazolyl, benzooxazolyl, quinolinyl,thiazolopyridinyl, pyrazolopyrimidinyl, etc.) optionally substitutedwith one or more R⁴. In some embodiments, R² is heteroaryl comprising a5-membered ring and 1-3 heteroatoms selected from N, O, and S,optionally substituted with one or more R⁴. In some embodiments, R² isheteroaryl comprising a 5-membered ring and 1-2 heteroatoms selectedfrom N, O, and S, optionally substituted with one or more R⁴. In someembodiments, R² is heteroaryl comprising a 6-membered ring and 1-3heteroatoms selected from N, O, and S, optionally substituted with oneor more R⁴. In some embodiments, R² is heteroaryl comprising a6-membered ring and 1-2 heteroatoms selected from N, O, and S,optionally substituted with one or more R⁴. In some embodiments, R² isheteroaryl comprising a 5-membered ring fused with a 6-membered ring and1-4 heteroatoms selected from N, O, and S, optionally substituted withone or more R⁴. In some embodiments, R² is selected from:

wherein each moiety is optionally substituted with one or more R⁴. Insome embodiments, R² is selected from:

wherein each moiety is optionally substituted with one or more R⁴. Insome embodiments, R² is

optionally substituted with one or more R⁴.

In some embodiments, at least one R⁴ is (C₁-C₄) alkyl, (C₁-C₄)haloalkyl, (C₁-C₄) alkoxy, (C₁-C₄) haloalkoxy, halogen, NO₂, OH, or CN.

In some embodiments, at least one R⁴ is (C₁-C₄) alkyl (e.g., methyl,ethyl, propyl, i-propyl, or butyl) optionally substituted with one ormore R⁵. In some embodiments, at least one R⁴ is methyl optionallysubstituted with one or more R⁵. In some embodiments, at least one R⁴ is(C₁-C₄) haloalkyl (e.g., CH₂F, CHF₂, or CF₃). In some embodiments, atleast one R⁴ is CF₃.

In some embodiments, at least one R⁴ is (C₁-C₄) alkoxy (e.g., methoxy,ethoxy, propoxy, or butoxy). In some embodiments, at least one R⁴ is(C₁-C₄) haloalkoxy (e.g., OCH₂F, OCHF₂, or OCF₃) In some embodiments, atleast one R⁴ is halogen (e.g., F, Cl, Br or I).

In some embodiments, at least one R⁴ is NO₂, OH, or CN.

In some embodiments, at least one R⁴ is C(O)R⁶ or C(O)OR⁶.

In some embodiments, at least one R⁴ is C(O)NR⁶R⁷ or NR⁶R⁷.

In some embodiments, at least one R⁴ is (C₃-C₇) cycloalkyl (e.g.,cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or cycloheptyl)optionally substituted with one or more R⁵.

In some embodiments, at least one R⁴ is heterocyclyl comprising a 5- to7-membered ring and 1-3 heteroatoms selected from N, O, and S (e.g.,pyrrolidinyl, pyrazolidinyl, imidazolidinyl, triazolidinyl,oxazolidinyl, isoxazolidinyl, oxadiazolidinyl, dioxazolidinyl,thiazolidinyl, isothiazolidinyl, thiadiazolidinyl, dithiazolidinyl,piperidinyl, hexahydropyridazinyl, hexahydropyrimidinyl, morpholinyl,dioxanyl, azepinyl, diazepinyl, etc.) optionally substituted with one ormore R⁵.

In some embodiments, at least one R⁴ is (C₆-C₁₀) aryl optionallysubstituted with one or more R⁵. In some embodiments, at least one R⁴ isphenyl optionally substituted with one or more R⁵.

In some embodiments, at least one R⁴ is heteroaryl comprising one or two5- to 7-membered rings and 1-4 heteroatoms selected from N, O, and S(e.g., pyrrolyl, pyrazolyl, imidazolyl, triazolyl, oxazolyl, isoxazolyl,oxadiazolyl, dioxazolyl, thiazolyl, isothiazolyl, thiadiazolyl,dithiazolyl, thiophenyl, pyridinyl, pyridazinyl, pyrimidinyl, triazinyl,benzothiazolyl, benzoimidazolyl, benzooxazolyl, quinolinyl, etc.)optionally substituted with one or more R⁵. In some embodiments, atleast one R⁴ is heteroaryl comprising a 5-membered ring (e.g., pyrrolyl,pyrazolyl, imidazolyl, triazolyl, oxazolyl, isoxazolyl, oxadiazolyl,dioxazolyl, thiazolyl, isothiazolyl, thiadiazolyl, dithiazolyl,thiophenyl, etc.) optionally substituted with one or more R⁵. In someembodiments, at least one R⁴ is heteroaryl comprising a 6-membered ring(e.g., pyridinyl, pyridazinyl, pyrimidinyl, triazinyl, etc.) optionallysubstituted with one or more R⁵.

In some embodiments, at least one R⁵ is (C₁-C₄) alkyl (e.g., methyl,ethyl, propyl, i-propyl, or butyl). In some embodiments, at least one R⁵is (C₁-C₄) haloalkyl (e.g., CH₂F, CHF₂, or CF₃).

In some embodiments, at least one R⁵ is (C₁-C₄) alkoxy (e.g., methoxy,ethoxy, propoxy, or butoxy). In some embodiments, at least one R⁵ is(C₁-C₄) haloalkoxy (e.g., OCH₂F, OCHF₂, or OCF₃).

In some embodiments, at least one R⁵ is halogen (e.g., F, Cl, Br or I).

In some embodiments, at least one R⁵ is NO₂, OH, or CN.

In some embodiments, at least one R⁶ is H. In some embodiments, at leastone R⁶ is (C₁-C₃) alkyl (e.g., methyl, ethyl, propyl, or i-propyl).

In some embodiments, at least one R⁷ is H. In some embodiments, at leastone R⁷ is (C₁-C₃) alkyl (e.g., methyl, ethyl, propyl, or i-propyl).

Any of the groups described herein for any of A, X, X², R¹, R², R³, R⁴,R⁵, R⁶, R⁷, R⁸, R⁹, n, and p can be combined with any of the groupsdescribed herein for one or more of the remainder of A, X, X², R¹, R²,R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, n, and p, and may further be combined withany of the groups described herein for the Linker.

For a Targeting Ligand of Formula TL-I:

-   -   (1) In one embodiment, X is N and A is phenyl.    -   (2) In one embodiment, X is CH and A is phenyl.    -   (3) In one embodiment, X is N, A is phenyl, and n is 0.    -   (4) In one embodiment, X is N, A is phenyl, n is 0, and R¹ is H.    -   (5) In one embodiment, X is N, A is phenyl, n is 1, and R¹ is H.    -   (6) In one embodiment, X is N, A is phenyl, n is 0, R¹ is H, and        R³ is

-   -   (7) In one embodiment, X is N, A is phenyl, n is 1, R¹ is H and        R³ is

-   -   (8) In one embodiment, X is N, A is phenyl, n is 0, R¹ is H, and        R³ is phenyl.    -   (9) In one embodiment, X is N, A is phenyl, n is 1, R¹ is H, and        R³ is phenyl.    -   (10) In one embodiment, X is N, A is phenyl, n is 0, R¹ is H, R³        is

-   -    and p is 1 or 2.    -   (11) In one embodiment, X is N, A is phenyl, n is 1, R¹ is H R³        is

-   -    and p is 1 or 2.    -   (12) In one embodiment, X is N, A is phenyl, n is 0, R¹ is H, R³        is

-   -    and R² is 5-membered heteroaryl comprising 1-4 heteroatoms        selected from N, O, and S, optionally substituted with one or        more R⁴.    -   (13) In one embodiment, X is N, A is phenyl, n is 1, R¹ is H, R³        is

-   -    and R² is 5-membered heteroaryl comprising 1-4 heteroatoms        selected from N, O, and S, optionally substituted with one or        more R⁴.    -   (14) In one embodiment, X is N, A is phenyl, n is 0, R₁ is H, R³        is

-   -    and R² is 5- or 6-membered heteroaryl comprising 1-4        heteroatoms selected from N, O, and S, optionally substituted        with one or more R⁴.    -   (15) In one embodiment, X is N, A is phenyl, n is 1, R¹ is H, R³        is

-   -    and R² is 5- or 6-membered heteroaryl comprising 1-4        heteroatoms selected from N, O, and S, optionally substituted        with one or more R⁴.    -   (16) In one embodiment, X is N, A is phenyl, n is 0, R¹ is H, R³        is

-   -    and R² is 5-membered heteroaryl comprising 1-4 heteroatoms        selected from N, O, and S.    -   (17) In one embodiment, X is N, A is phenyl, n is 1, R¹ is H, R³        is

-   -    and R² is 5-membered heteroaryl comprising 1-4 heteroatoms        selected from N, O, and S.    -   (18) In one embodiment, X is N, A is phenyl, n is 0, R¹ is H, R³        is

-   -    and R² is 5- or 6-membered heteroaryl comprising 1-4        heteroatoms selected from N, O, and S.    -   (19) In one embodiment, X is N, A is phenyl, n is 1, R¹ is H, R³        is

-   -    and R² is 5- or 6-membered heteroaryl comprising 1-4        heteroatoms selected from N, O, and

S.

-   -   (20) In one embodiment, X is N, A is phenyl, n is 0, R¹ is H, R³        is

-   -    R² is 5-membered heteroaryl comprising 1-4 heteroatoms selected        from N, O, and S, and at least one R⁹ is NO₂, NH₂, OH, or F.    -   (21) In one embodiment, X is N, A is phenyl, n is 1, R¹ is H, R³        is

-   -    R² is 5-membered heteroaryl comprising 1-4 heteroatoms selected        from N, O, and S, and at least one R⁹ is NO₂, NH₂, OH, or F.    -   (22) In one embodiment, X is N, A is phenyl, n is 0, R¹ is H, R³        is

-   -    R² is 5-membered heteroaryl comprising 1-4 heteroatoms selected        from N, O, and S, optionally substituted with one or more R⁴,        and at least one R⁹ is NO₂, NH₂, OH, or F.    -   (23) In one embodiment, X is N, A is phenyl, n is 1, R¹ is H, R³        is

-   -    R² is 5-membered heteroaryl comprising 1-4 heteroatoms selected        from N, O, and S, optionally substituted with one or more R⁴,        and at least one R⁹ is NO₂, NH₂, OH, or F.

In one embodiment, the compound of Formula TL-I is of Formula TL-Ia orTL-Ib:

wherein A, X, R², R⁹, and p are each as defined above in Formula TL-I.

In one embodiment, A is phenyl.

In one embodiment, X is N.

In one embodiment, at least one R⁹ is F or OH.

In one embodiment, p is 0, 1, or 2.

In one embodiment, R² is heteroaryl comprising one or two 5- to7-membered rings and 1-4 heteroatoms selected from N, O, and S,optionally substituted with one or more R⁴. In another embodiment, R² isheteroaryl comprising one 5- to 7-membered ring and 1-4 heteroatomsselected from N, O, and S, optionally substituted with one or more R⁴.In another embodiment, R² is 5-membered heteroaryl comprising and 1-4heteroatoms selected from N, O, and S, optionally substituted with oneor more R⁴. In another embodiment, R² is 5-membered heteroarylcomprising and 1-2 heteroatoms selected from N, O, and S, optionallysubstituted with one or more R⁴.

A, X, R², R⁹, and p can each be selected from any of the groups andcombined as described above in Formula TL-I.

In another embodiment, the compound of Formula TL-I is of Formula TL-Icor TL-Id:

wherein A, X, R², R⁹, and p are each as defined above in Formula TL-I.

In one embodiment, A is phenyl.

In one embodiment, X is N.

In one embodiment, at least one R⁹ is F or OH.

In one embodiment, p is 0, 1, or 2.

In one embodiment, R² is heteroaryl comprising one or two 5- to7-membered rings and 1-4 heteroatoms selected from N, O, and S,optionally substituted with one or more R⁴. In another embodiment, R² isheteroaryl comprising one 5- to 7-membered ring and 1-4 heteroatomsselected from N, O, and S, optionally substituted with one or more R⁴.In another embodiment, R² is 5-membered heteroaryl comprising and 1-4heteroatoms selected from N, O, and S, optionally substituted with oneor more R⁴. In another embodiment, R² is 5-membered heteroarylcomprising and 1-2 heteroatoms selected from N, O, and S, optionallysubstituted with one or more R⁴.

A, X, R², R⁹, and p can each be selected from any of the groups andcombined as described above in Formula TL-I.

In another embodiment, the compound of Formula TL-I is of Formula TL-Ieor TL-If:

wherein R², R⁹, and p are each as defined above in Formula TL-I.

In one embodiment, at least one R⁹ is F or OH.

In one embodiment, p is 0, 1 or 2.

In one embodiment, R² is heteroaryl comprising one or two 5- to7-membered rings and 1-4 heteroatoms selected from N, O, and S,optionally substituted with one or more R⁴. In another embodiment, R² isheteroaryl comprising one 5- to 7-membered ring and 1-4 heteroatomsselected from N, O, and S, optionally substituted with one or more R⁴.In another embodiment, R² is 5-membered heteroaryl comprising and 1-4heteroatoms selected from N, O, and S, optionally substituted with oneor more R⁴.

R², R⁹, and p can each be selected from any of the groups and combinedas described above in Formula TL-I.

In one embodiment, the compound of Formula TL-I is a compound of aformula selected from the group consisting of:

In one embodiment, the compound of Formula TL-I is a compound of aformula selected from the group consisting of:

Targeting Ligand (TL) (or target protein moiety or target protein ligandor ligand) is a small molecule which is capable of binding to a targetprotein of interest, such as EGFR or a mutant thereof.

Degron

The Degron serves to link a targeted protein, through a Linker and aTargeting Ligand, to a ubiquitin ligase for proteosomal degradation. Inone embodiment, the Degron is capable of binding to a ubiquitin ligase,such as an E3 ubiquitin ligase. In one embodiment, the Degron is capableof binding to cereblon.

In one embodiment, the Degron is of Formula D1:

or an enantiomer, diastereomer, or stereoisomer thereof, wherein:

Y is a bond, (CH₂)₁₋₆, (CH₂)₀₋₆—O, (CH₂)₀₋₆—C(O)NR¹¹, (CH₂)₀₋₆—NR¹¹C(O),(CH₂)₀₋₆—NH, or (CH₂)₀₋₆—NR¹²;

Z¹ is C(O) or C(R¹³)₂;

R¹¹ is H or C₁-C₆ alkyl;

R¹² is C₁-C₆ alkyl or C(O)—C₁-C₆ alkyl;

each R¹³ is independently H or C₁-C₃ alkyl;

each R¹⁴ is independently C₁-C₃ alkyl;

R¹⁵ is H, deuterium, C₁-C₃ alkyl, F, or Cl;

each R¹⁶ is independently halogen, OH, C₁-C₆ alkyl, or C₁-C₆ alkoxy;

q is 0, 1, or 2; and

v is 0, 1, 2, or 3,

wherein the Degron is covalently bonded to the Linker via

In one embodiment, Z¹ is C(O).

In one embodiment, Z¹ is C(R¹³)₂; and each R¹³ is H. In one embodiment,Z¹ is C(R¹³)₂; and one of R¹³ is H, and the other is C₁-C₃ alkylselected from methyl, ethyl, and propyl. In one embodiment, Z¹ isC(R¹³)₂; and each R¹³ is independently selected from methyl, ethyl, andpropyl.

In one embodiment, Y is a bond.

In one embodiment, Y is (CH₂)₁, (CH₂)₂, (CH₂)₃, (CH₂)₄, (CH₂)₅, or(CH₂)₆. In one embodiment, Y is (CH₂)₁, (CH₂)₂, or (CH₂)₃. In oneembodiment, Y is (CH₂)₁ or (CH₂)₂.

In one embodiment, Y is O, CH₂—O, (CH₂)₂—O, (CH₂)₃—O, (CH₂)₄—O,(CH₂)₅—O, or (CH₂)₆—O. In one embodiment, Y is O, CH₂—O, (CH₂)₂—O, or(CH₂)₃₋₀. In one embodiment, Y is O or CH₂—O. In one embodiment, Y is O.

In one embodiment, Y is C(O)NR¹¹, CH₂—C(O)NR¹¹, (CH₂)₂—C(O)NR¹¹,(CH₂)₃—C(O)NR¹¹, (CH₂)₄—C(O)NR¹¹, (CH₂)₅—C(O)NR¹¹, or (CH₂)₆—C(O)NR¹¹.In one embodiment, Y is C(O)NR¹¹, CH₂—C(O)NR¹¹, (CH₂)₂—C(O)NR¹¹, or(CH₂)₃—C(O)NR¹¹. In one embodiment, Y is C(O)NR¹¹ or CH₂—C(O)NR¹¹. Inone embodiment, Y is C(O)NR¹¹.

In one embodiment, Y is NR¹¹C(O), CH₂—NR¹¹C(O), (CH₂)₂—NR¹¹C(O),(CH₂)₃—NR¹¹C(O), (CH₂)₄—NR¹¹C(O), (CH₂)₅—NR¹¹C(O), or (CH₂)₆—NR¹¹C(O).In one embodiment, Y is NR¹¹C(O), CH₂—NR¹¹C(O), (CH₂)₂—NR¹¹C(O), or(CH₂)₃—NR¹¹C(O). In one embodiment, Y is NR¹¹C(O) or CH₂—NR¹¹C(O). Inone embodiment, Y is NR¹¹C(O).

In one embodiment, R¹¹ is H. In one embodiment, R¹¹ is selected frommethyl, ethyl, propyl, butyl, i-butyl, t-butyl, pentyl, i-pentyl, andhexyl. In one embodiment, R¹¹ is C₁-C₃ alkyl selected from methyl,ethyl, and propyl.

In one embodiment, Y is NH, CH₂—NH, (CH₂)₂—NH, (CH₂)₃—NH, (CH₂)₄—NH,(CH₂)₅—NH, or (CH₂)₆—NH. In one embodiment, Y is NH, CH₂—NH, (CH₂)₂—NH,or (CH₂)₃—NH. In one embodiment, Y is NH or CH₂—NH. In one embodiment, Yis NH.

In one embodiment, Y is NR¹², CH₂—NR¹², (CH₂)₂—NR¹², (CH₂)₃—NR¹²,(CH₂)₄—NR¹², (CH₂)₅—NR¹², or (CH₂)₆—NR¹². In one embodiment, Y is NR¹²,CH₂—NR¹², (CH₂)₂—NR¹², or (CH₂)₃—NR¹². In one embodiment, Y is NR¹² orCH₂—NR¹². In one embodiment, Y is NR¹².

In one embodiment, R¹² is selected from methyl, ethyl, propyl, butyl,i-butyl, t-butyl, pentyl, i-pentyl, and hexyl. In one embodiment, R¹² isC₁-C₃ alkyl selected from methyl, ethyl, and propyl.

In one embodiment, R¹² is selected from C(O)-methyl, C(O)-ethyl,C(O)-propyl, C(O)-butyl, C(O)-i-butyl, C(O)-t-butyl, C(O)-pentyl,C(O)-i-pentyl, and C(O)-hexyl. In one embodiment, R¹² is C(O)—C₁-C₃alkyl selected from C(O)-methyl, C(O)-ethyl, and C(O)-propyl.

In one embodiment, R¹³ is H.

In one embodiment, R¹³ is C₁-C₃ alkyl selected from methyl, ethyl, andpropyl. In one embodiment, R¹³ is methyl.

In one embodiment, q is 0.

In one embodiment, q is 1.

In one embodiment, q is 2.

In one embodiment, each R¹⁴ is independently C₁-C₃ alkyl selected frommethyl, ethyl, and propyl.

In one embodiment, v is 0.

In one embodiment, v is 1.

In one embodiment, v is 2.

In one embodiment, v is 3.

In one embodiment, each R¹⁶ is independently selected from halogen(e.g., F, Cl, Br, and I), OH, C₁-C₆ alkyl (e.g., methyl, ethyl, propyl,butyl, i-butyl, t-butyl, pentyl, i-pentyl, and hexyl), and C₁-C₆ alkoxy(e.g., methoxy, ethoxy, propoxy, butoxy, i-butoxy, t-butoxy, andpentoxy). In a further embodiment, each R¹⁶ is independently selectedfrom F, Cl, OH, methyl, ethyl, propyl, butyl, i-butyl, t-butyl, methoxy,and ethoxy.

In one embodiment, R¹⁵ is H, deuterium, or C₁-C₃ alkyl. In anotherembodiment, R¹⁵ is H or C₁-C₃ alkyl. In a further embodiment, R¹⁵ is inthe (S) or (R) configuration. In a further embodiment, R¹⁵ is in the (S)configuration. In one embodiment, the compound comprises a racemicmixture of (S)—R¹⁵ and (R)—R¹⁵.

In one embodiment, R¹⁵ is H.

In one embodiment, R¹⁵ is deuterium.

In one embodiment, R¹⁵ is C₁-C₃ alkyl selected from methyl, ethyl, andpropyl. In one embodiment, R¹⁵ is methyl.

In one embodiment, R¹⁵ is F or Cl. In a further embodiment, R¹⁵ is inthe (S) or (R) configuration. In a further embodiment, R¹⁵ is in the (R)configuration. In one embodiment, the compound comprises a racemicmixture of (S)—R¹⁵ and (R)—R¹⁵. In one embodiment, R¹⁵ is F.

Any of the groups described herein for any of Y, Z¹, R¹¹, R¹², R¹³, R¹⁴,R¹⁵, R¹⁶, q, and v can be combined with any of the groups describedherein for one or more of the remainder of Y, Z¹, R¹¹, R¹², R¹³, R¹⁴,R¹⁵, R¹⁶, q, and v, and may further be combined with any of the groupsdescribed herein for the Linker.

For a Degron of Formula D1:

-   -   (1) In one embodiment, Z¹ is C(O) and Y is a bond.    -   (2) In one embodiment, Z¹ is C(O) and Y is (CH₂)₀₋₆—O. In a        further embodiment, Y is O.    -   (3) In one embodiment, Z¹ is C(O); Y is a bond; and q and v are        each 0.    -   (4) In one embodiment, Z¹ is C(O); Y is a bond; and R¹³ is H.    -   (5) In one embodiment, Z¹ is C(O); Y is a bond; and R¹⁵ is H.    -   (6) In one embodiment, Z¹ is C(O); Y is a bond; and R¹³ is H;        and R¹⁵ is H.    -   (7) In one embodiment, Z¹ is C(O); Y is (CH₂)₀₋₆—O; and R¹³        is H. In a further embodiment, Y is O.    -   (8) In one embodiment, Z¹ is C(O); Y is (CH₂)₀₋₆—O; and R¹⁵        is H. In a further embodiment, Y is O.    -   (9) In one embodiment, Z¹ is C(O); Y is (CH₂)₀₋₆—O; R¹³ is H;        and R¹⁵ is H. In a further embodiment, Y is O.    -   (10) In one embodiment, Z¹ is C(O); Y is (CH₂)₀₋₆—NH; and R¹³        is H. In a further embodiment, Y is NH.    -   (11) In one embodiment, Z¹ is C(O); Y is (CH₂)₀₋₆—NH; and R¹⁵        is H. In a further embodiment, Y is NH.    -   (12) In one embodiment, Z¹ is C(O); Y is (CH₂)₀₋₆—NH; R¹³ is H;        and R¹⁵ is H. In a further embodiment, Y is NH.    -   (13) In one embodiment, q and v are each 0; and Y, Z¹, R¹³, and        R¹⁵ are each as defined in any of (1)-(12).

In one embodiment, the Degron is of Formula D1a-D1d:

or an enantiomer, diastereomer, or stereoisomer thereof, wherein R¹⁴,R¹⁶, q, and v are each as defined above in Formula D1, and can beselected from any moieties or combinations thereof described above.

In one embodiment, the Degron is of Formula D2:

or an enantiomer, diastereomer, or stereoisomer thereof, wherein:

each R¹⁷ is independently C₁-C₃ alkyl;

q′ is 0, 1, 2, 3 or 4; and

R¹⁸ is H or C₁-C₃ alkyl,

wherein the Degron is covalently bonded to another moiety via

In one embodiment, the Degron is covalently bonded to a Linker via

In one embodiment, the Degron is covalently bonded to a compound via

In one embodiment, q′ is 0.

In one embodiment, q′ is 1.

In one embodiment, q′ is 2.

In one embodiment, q′ is 3.

In one embodiment, each R¹⁷ is independently C₁-C₃ alkyl selected frommethyl, ethyl, and propyl.

In one embodiment, R⁸ is methyl, ethyl, or propyl. In one embodiment, R⁸is methyl.

In one embodiment, the Degron is of Formula D2a or D2b:

In an alternative embodiment, the Degron is of Formula D3:

or an enantiomer, diastereomer, or stereoisomer thereof, wherein:

-   -   each R¹⁷ is independently C₁-C₃ alkyl;    -   q′ is 0, 1, 2, 3 or 4;

R¹⁸ is H or C₁-C₃ alkyl; and

R¹⁹ is C₁-C₃ alkyl,

wherein the Degron is covalently bonded to another moiety via

In one embodiment, the Degron is covalently bonded to a Linker via

In one embodiment, the Degron is covalently bonded to a compound via

In one embodiment, q′ is 0.

In one embodiment, q′ is 1.

In one embodiment, q′ is 2.

In one embodiment, q′ is 3.

In one embodiment, each R¹⁷ is independently C₁-C₃ alkyl selected frommethyl, ethyl, and propyl.

In one embodiment, R⁸ is methyl, ethyl, or propyl. In one embodiment, R⁸is methyl.

In one embodiment, R¹⁹ is methyl, ethyl, or propyl. In one embodiment,R¹⁹ is methyl.

In one embodiment, the Degron is of Formula D3a or D3b:

Linker

The Linker is a bond or a carbon chain that serves to link a TargetingLigand with a Degron. In one embodiment, the carbon chain optionallycomprises one, two, three, or more heteroatoms selected from N, O, andS. In one embodiment, the carbon chain comprises only saturated chaincarbon atoms. In one embodiment, the carbon chain optionally comprisestwo or more unsaturated chain carbon atoms (e.g., C═C or C≡C). In oneembodiment, one or more chain carbon atoms in the carbon chain areoptionally substituted with one or more substituents (e.g., oxo, C₁-C₆alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₃ alkoxy, OH, halogen, NH₂,NH(C₁-C₃ alkyl), N(C₁-C₃ alkyl)₂, CN, C₃-C₈ cycloalkyl, heterocyclyl,phenyl, and heteroaryl).

In one embodiment, the Linker comprises at least 5 chain atoms (e.g., C,O, N, and S). In one embodiment, the Linker comprises less than 25 chainatoms (e.g., C, O, N, and S). In one embodiment, the Linker comprisesless than 20 chain atoms (e.g., C, O, N, and S). In one embodiment, theLinker comprises 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,or 20, chain atoms (e.g., C, O, N, and S). In one embodiment, the Linkercomprises 5, 7, 9, 11, 13, 15, 17, or 19 chain atoms (e.g., C, O, N, andS). In one embodiment, the Linker comprises 5, 7, 9, or 11 chain atoms(e.g., C, O, N, and S). In one embodiment, the Linker comprises 11, 13,15, 17, or 19 chain atoms (e.g., C, O, N, and S). In one embodiment, theLinker comprises 6, 8, 10, 12, 14, 16, 18, or 20 chain atoms (e.g., C,O, N, and S). In one embodiment, the Linker comprises 6, 8, 10, or 12chain atoms (e.g., C, O, N, and S). In one embodiment, the Linkercomprises 12, 14, 16, 18, or 20 chain atoms (e.g., C, O, N, and S).

In one embodiment, the Linker comprises from 9 to 19 chain atoms (e.g.,C, O, N, and S).

In one embodiment, the Linker is a carbon chain optionally substitutedwith non-bulky substituents (e.g., oxo, C₁-C₆ alkyl, C₂-C₆ alkenyl,C₂-C₆ alkynyl, C₁-C₃ alkoxy, OH, halogen, NH₂, NH(C₁-C₃ alkyl), N(C₁-C₃alkyl)₂, and CN). In one embodiment, the non-bulky substitution islocated on the chain carbon atom proximal to the Degron (i.e., thecarbon atom is separated from the carbon atom to which the Degron isbonded by at least 3, 4, or 5 chain atoms in the Linker). In oneembodiment, the non-bulky substitution is located on the chain carbonatom proximal to the Targeting Ligand (i.e., the carbon atom isseparated from the carbon atom to which the Degron is bonded by at least3, 4, or 5 chain atoms in the Linker).

In one embodiment, the Linker is of Formula L0:

or an enantiomer, diastereomer, or stereoisomer thereof, wherein

p1 is an integer selected from 0 to 12;

-   -   p2 is an integer selected from 0 to 12;    -   p3 is an integer selected from 0 to 6;    -   each W is independently absent, CH₂, O, S, NH, or NR¹⁰;

Z is absent, CH₂, O, NH, NR¹⁰, C(O)(CH₂)₀₋₃, or NHC(O)(CH₂)₀₋₃;

each R¹⁰ is independently H or C₁-C₃ alkyl; and

Q is absent or CH₂C(O)NH,

wherein the Linker is covalently bonded to the Degron via the

next to Q, and covalently bonded to the Targeting Ligand via the

next to Z.

In an alternative embodiment, the Linker is of Formula L0′:

or an enantiomer, diastereomer, or stereoisomer thereof, wherein

p1 is an integer selected from 0 to 12;

p2 is an integer selected from 0 to 12;

p3 is an integer selected from 0 to 6;

each W is independently absent, CH₂, O, S, NH, or NR¹⁰;

Z is absent, CH₂, O, NH, NR¹⁰, C(O)(CH₂)₀₋₃, or NHC(O)(CH₂)₀₋₃;

each R¹⁰ is independently H or C₁-C₃ alkyl; and

Q is a heterocycle,

wherein the Linker is covalently bonded to the Degron via the

next to Q, and covalently bonded to the Targeting Ligand via the

next to Z.

In one embodiment, the total number of chain atoms in the Linker is lessthan 30. In a further embodiment, the total number of chain atoms in theLinker is less than 20.

For a Linker of Formula L0:

In one embodiment, p1 is an integer selected from 0 to 10.

In one embodiment, p1 is an integer selected from 1 to 10.

In one embodiment, p1 is selected from 1, 2, 3, 4, 5, and 6.

In one embodiment, p1 is 0, 1, 3, or 5.

In one embodiment, p1 is 0, 1, 2, or 3.

In one embodiment, p1 is 1.

In one embodiment, p1 is 3.

In one embodiment, p1 is 5.

In one embodiment, p2 is an integer selected from 0 to 10.

In one embodiment, p2 is selected from 0, 1, 2, 3, 4, 5, and 6.

In one embodiment, p2 is 0, 1, 2, or 3.

In one embodiment, p2 is 0.

In one embodiment, p2 is 1.

In one embodiment, p3 is an integer selected from 0 to 5.

In one embodiment, p3 is 1, 2, 3, 4, or 5.

In one embodiment, p3 is 1, 2, or 3.

In one embodiment, p3 is 0.

In one embodiment, p3 is 2 or 3.

In one embodiment, p3 is 2.

In one embodiment, at least one W is CH₂.

In one embodiment, at least one W is O.

In one embodiment, at least one W is S.

In one embodiment, at least one W is NH.

In one embodiment, at least one W is NR¹⁰; and R¹⁰ is C₁-C₃ alkylselected from methyl, ethyl, and propyl.

In one embodiment, each W is O.

In one embodiment, Q is absent.

In one embodiment, Q is CH₂C(O)NH.

In one embodiment, Z is C(O), C(O)(CH₂), C(O)(CH₂)₂, or C(O)(CH₂)₃. Inone embodiment, Z is C(O).

In one embodiment, Z is NHC(O), NHC(O)(CH₂), NHC(O)(CH₂)₂, orNHC(O)(CH₂)₃. In one embodiment, Z is NHC(O)(CH₂).

In one embodiment, Z is absent.

In one embodiment, Z is CH₂.

In one embodiment, Z is O.

In one embodiment, Z is NH.

In one embodiment, Z is NR¹⁰; and R¹⁰ is C₁-C₃ alkyl selected frommethyl, ethyl, and propyl.

In one embodiment, Z is part of the Targeting Ligand that is bonded tothe Linker, namely, Z is formed from reacting a functional group of theTargeting Ligand with the Linker.

In one embodiment, the Linker-Targeting Ligand (TL) has the structureselected from Table L1:

wherein Q, Z, and p1 are each as described above, and TL is a TargetingLigand.

In one embodiment, p1 is 0, 1, 2, 3, 4, or 5. In one embodiment, p1 is0. In one embodiment, p1 is 1, 3, or 5. In one embodiment, p1 is 1. Inone embodiment, p1 is 3. In one embodiment, p1 is 5.

In one embodiment, Q is CH₂C(O)NH.

In one embodiment, Q is absent.

In one embodiment, Z is C(O)(CH₂)₀₋₃. In one embodiment, Z is C(O). Inone embodiment, Z is NHC(O)(CH₂)₀₋₃. In one embodiment, Z isNHC(O)(CH₂).

In one embodiment, Q is CH₂C(O)NH, and Z is NHC(O)(CH₂).

In one embodiment, Q is CH₂C(O)NH, and Z is C(O).

In one embodiment, Q is absent, and Z is NHC(O)(CH₂).

In one embodiment, Q is absent, and Z is C(O).

For a Linker of Formula L0′:

In one embodiment, p1 is an integer selected from 0 to 10.

In one embodiment, p1 is an integer selected from 1 to 10.

In one embodiment, p1 is selected from 1, 2, 3, 4, 5, and 6.

In one embodiment, p1 is 0, 1, 3, or 5.

In one embodiment, p1 is 0, 1, 2, or 3.

In one embodiment, p1 is 1.

In one embodiment, p1 is 3.

In one embodiment, p1 is 5.

In one embodiment, p2 is an integer selected from 0 to 10.

In one embodiment, p2 is selected from 0, 1, 2, 3, 4, 5, and 6.

In one embodiment, p2 is 0, 1, 2, or 3.

In one embodiment, p2 is 0.

In one embodiment, p2 is 1.

In one embodiment, p3 is an integer selected from 0 to 5.

In one embodiment, p3 is 1, 2, 3, 4, or 5.

In one embodiment, p3 is 1, 2, or 3.

In one embodiment, p3 is 0.

In one embodiment, p3 is 2 or 3.

In one embodiment, p3 is 2.

In one embodiment, at least one W is CH₂.

In one embodiment, at least one W is O.

In one embodiment, at least one W is S.

In one embodiment, at least one W is NH.

In one embodiment, at least one W is NR¹⁰; and R¹⁰ is C₁-C₃ alkylselected from methyl, ethyl, and propyl.

In one embodiment, each W is O.

In one embodiment, Q is a heterocycle.

In one embodiment, Q is azetidine.

In one embodiment, Z is C(O), C(O)(CH₂), C(O)(CH₂)₂, or C(O)(CH₂)₃. Inone embodiment, Z is C(O).

In one embodiment, Z is NHC(O), NHC(O)(CH₂), NHC(O)(CH₂)₂, orNHC(O)(CH₂)₃. In one embodiment, Z is NHC(O)(CH₂).

In one embodiment, Z is absent.

In one embodiment, Z is CH₂.

In one embodiment, Z is O.

In one embodiment, Z is NH.

In one embodiment, Z is NR¹⁰; and R¹⁰ is C₁-C₃ alkyl selected frommethyl, ethyl, and propyl.

In one embodiment, Z is part of the Targeting Ligand that is bonded tothe Linker, namely, Z is formed from reacting a functional group of theTargeting Ligand with the Linker.

In an alternative embodiment, the Linker-Targeting Ligand (TL) has thestructure selected from Table L2:

In one embodiment, p1 is 0, 1, 2, 3, 4, or 5. In one embodiment, p1 is0. In one embodiment, p1 is 1, 3, or 5. In one embodiment, p1 is 1. Inone embodiment, p1 is 3. In one embodiment, p1 is 5.

In one embodiment, Q is a heterocycle.

In one embodiment, Q is azetidine.

In one embodiment, Z is C(O)(CH₂)₀₋₃. In one embodiment, Z is C(O). Inone embodiment, Z is NHC(O)(CH₂)₀₋₃. In one embodiment, Z isNHC(O)(CH₂).

In one embodiment, Q is a heterocycle, and Z is NHC(O)(CH₂).

In one embodiment, Q is a heterocycle, and Z is C(O).

In one embodiment, Q is an azetidine, and Z is NHC(O)(CH₂).

In one embodiment, Q is an azetidine, and Z is C(O).

In one embodiment, the Linker-Targeting Ligand has the structureselected from L2, L3, L7, L8, L12, L13, L17, L18, L22, L23, L27, L28,L32, L33, L37, L38, and L47. In one embodiment, the Linker-TargetingLigand has the structure selected from L18, L23, L32, and L47.

Any one of the Degrons described herein can be covalently bound to anyone of the Linkers described herein. Any one of the Targeting Ligandsdescribed herein can be covalently bound to any one of the Linkersdescribed herein.

In one embodiment, the present application includes the Degron-Linker(DL), wherein the Degron is of Formula D1, and the Linker is selectedfrom L1-L48. In one embodiment, the present application includes theDegron-Linker (DL), wherein the Degron is one of Formula D1a-D1d, andthe Linker is selected from L1-L48. In one embodiment, the Degron is oneof Formula D1a-D1d, and the Linker is selected from L18, L23, L32, andL47. In one embodiment, the Degron is of Formula D1a or D1b, and theLinker is L18 or L23. In one embodiment, the Degron is of Formula D1c orD1d, and the Linker is L32.

In one embodiment, the Linker is designed and optimized based on SAR(structure-activity relationship) and X-ray crystallography of theTargeting Ligand with regard to the location of attachment for theLinker.

In one embodiment, the optimal Linker length and composition vary by theTargeting Ligand and can be estimated based upon X-ray structure of theTargeting Ligand bound to its target. Linker length and composition canbe also modified to modulate metabolic stability and pharmacokinetic(PK) and pharmacodynamics (PD) parameters.

Some embodiments of present application relate to the bifunctionalcompounds having the following structures in Table A1 and Table A2:

TABLE A1 Cmpd No. Structure   I-1

I-2

I-3

I-4

I-5

I-6

TABLE A2 Cmpd No. Structure I-7 

I-8 

I-9 

I-10

I-11

I-12

I-13

I-14

I-15

I-16

I-17

I-18

I-19

I-20

I-21

I-22

I-23

I-24

I-25

I-26

I-27

I-28

I-29

I-30

I-31

I-32

I-33

I-34

I-35

Some of the foregoing compounds can include one or more asymmetriccenters, and thus can exist in various isomeric forms. In oneembodiment, the compounds exist as stereoisomers. In one embodiment, thecompounds exist as diastereomers. Accordingly, compounds of theapplication may be in the form of an individual enantiomer, diastereomeror geometric isomer, or may be in the form of a mixture ofstereoisomers. In one embodiment, the compounds of the application areenantiopure compounds. In another embodiment, mixtures of stereoisomersor diastereomers are provided.

Furthermore, certain compounds, as described herein, may have one ormore double bonds that can exist as either the Z or E isomer, unlessotherwise indicated. The application additionally encompasses thecompounds as individual Z/E isomers substantially free of other E/Zisomers and alternatively, as mixtures of various isomers.

In one embodiment, the present application provides compounds thattarget proteins, such as EGFR for degradation, which have numerousadvantages over inhibitors of protein function (e.g., kinase activity)and can a) overcome resistance in certain cases; b) prolong the kineticsof drug effect by destroying the protein, thus requiring resynthesis ofthe protein even after the compound has been metabolized; c) target allfunctions of a protein at once rather than a specific catalytic activityor binding event; d) expand the number of drug targets by including allproteins that a ligand can be developed for, rather than proteins whoseactivity (e.g., kinase activity) can be affected by a small moleculeinhibitor, antagonist or agonist; and e) have increased potency comparedto inhibitors due to the possibility of the small molecule actingcatalytically.

Some embodiments of the present application relate to degradation orloss of 30% to 100% of the target protein. Some embodiments relate tothe loss of 50-100% of the target protein. Other embodiments relate tothe loss of 75-95% of the targeted protein.

A bifunctional compound of Formula X, Formula Y, Formula Z, Formula A,Formula B, Formula C, or Formula D is capable of modulating ordecreasing the amount of a targeted protein (e.g., EGFR). A bifunctionalcompound of Formula X, Formula Y, Formula Z, Formula A, Formula B,Formula C, or Formula D is also capable of degrading a targeted protein(e.g., EGFR) through the UPP pathway. A bifunctional compound of FormulaX, Formula Y, Formula Z, Formula A, Formula B, Formula C, or Formula Dis also capable of preventing EGFR dimer formation. Accordingly, abifunctional compound of the present application (e.g., a bifunctionalcompound of any of the formulae described herein, or selected from anybifunctional compounds described herein) is also capable of treating orpreventing a disease or disorder in which EGFR plays a role (e.g.,through EGFR dimer formation) or in which EGFR is deregulated (e.g.,overexpressed).

In some embodiments, the bifunctional compounds of the presentapplication bind to an EGFR or a mutant thereof covalently. In otherembodiments, the bifunctional compounds of the present application bindto an EGFR or a mutant thereof non-covalently.

In some embodiments, the bifunctional compounds of Formula X, Formula Y,Formula Z, Formula A, Formula B, Formula C, or Formula D are capable ofinhibiting or decreasing the activity of EGFR containing one or moremutations. In some embodiments, the mutant EGFR contains one or moremutations selected from T790M, L718Q, L844V, V948R, L858R, I941R, C797S,and Del. In other embodiments, the mutant EGFR contains a combination ofmutations, wherein the combination is selected from Del/L718Q,Del/L844V, Del/T790M, Del/T790M/L718Q, Del/T790M/L844V, L858R/L718Q,L858R/L844V, L858R/T790M, L858R/T790M/I941R, Del/T790M, Del/T790M/C797S,L858R/T790M/C797S, and L858R/T790M/L718Q. In other embodiments, themutant EGFR contains a combination of mutations, wherein the combinationis selected from Del/L844V, L858R/L844V, L858R/T790M, L858R/T790M/I941R,L858R/T790M/C797S, Del/T790M, Del/T790M, Del/T790M/C797S, andL858R/T790M. In other embodiments, the mutant EGFR contains acombination of mutations, wherein the combination is selected fromL858R/T790M, L858R/T790M/I941R, L858R/T790M/C797S, Del/T790M,Del/T790M/C797S, and L858R/T790M.

Modulation of EGFR through UPP-mediated degradation by a bifunctionalcompound of Formula X, Formula Y, Formula Z, Formula A, Formula B,Formula C, or Formula D provides a novel approach to the treatment,prevention, or amelioration of diseases or disorders in which EGFR playsa role including, but not limited to, cancer and metastasis,inflammation, arthritis, systemic lupus erthematosus, skin-relateddisorders, pulmonary disorders, cardiovascular disease, ischemia,neurodegenerative disorders, liver disease, gastrointestinal disorders,viral and bacterial infections, central nervous system disorders,Alzheimer's disease, Parkinson's disease, Huntington's disease,amyotrophic lateral sclerosis, spinal cord injury, and peripheralneuropathy. Further, modulation of EGFR through UPP-mediated degradationby a bifunctional compound of the application, such as those describedherein, also provides a new paradigm for treating, preventing, orameliorating diseases or disorders in which EGFR is deregulated.

In one embodiment, a bifunctional compound of the Formula X, Formula Y,Formula Z, Formula A, Formula B, Formula C, or Formula D is moreefficacious in treating a disease or condition (e.g., cancer) than, oris capable of treating a disease or condition resistant to, theTargeting Ligand, when the Targeting Ligand is administered alone (i.e.,not bonded to a Linker and a Degron). In one embodiment, a bifunctionalcompound of the present application (e.g., a bifunctional compound ofany of the formulae described herein, or selected from any bifunctionalcompounds described herein) is capable of modulating (e.g., decreasing)the amount of EGFR, and thus is useful in treating a disease orcondition (e.g., cancer) in which the EGFR plays a role.

In one embodiment, the bifunctional compound of Formula X, Formula Y,Formula Z, Formula A, Formula B, Formula C, or Formula D is moreefficacious in treating a disease or condition or is capable of treatinga disease or condition resistant to the Targeting Ligand, when theTargeting Ligand is administered alone (i.e., not bonded to a Linker anda Degron), is more potent in inhibiting the growth of cells (e.g.,cancer cells) or decreasing the viability of cells (e.g., cancer cells),than the Targeting Ligand, when the Targeting Ligand is administeredalone (i.e., not bonded to a Linker and a Degron). In one embodiment,the bifunctional compound inhibits the growth of cells (e.g., cancercells) or decreases the viability of cells (e.g., cancer cells) at anIC₅₀ that is lower than the IC₅₀ of the Targeting Ligand (when theTargeting Ligand is administered alone (i.e., not bonded to a Linker anda Degron)) for inhibiting the growth or decreasing the viability of thecells. In one embodiment, the IC₅₀ of the bifunctional compound is atmost 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 8%, 5%, 4%, 3%, 2%,1%, 0.8%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% of the IC₅₀ of the TargetingLigand. In one embodiment, the IC₅₀ of the bifunctional compound is atmost 50%, 40%, 30%, 20%, 10%, 8%, 5%, 4%, 3%, 2%, 1%, 0.8%, 0.5%, 0.4%,0.3%, 0.2%, or 0.1% of the IC₅₀ of the Targeting Ligand. In oneembodiment, the IC₅₀ of the bifunctional compound is at most 30%, 20%,10%, 8%, 5% 4%, 3%, 2%, 1%, 0.8%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% of theIC₅₀ of the Targeting Ligand. In one embodiment, the IC₅₀ of thebifunctional compound is at most 10%, 8%, 5% 4%, 3%, 2%, 1%, 0.8%, 0.5%,0.4%, 0.3%, 0.2%, or 0.1% of the IC₅₀ of the Targeting Ligand. In oneembodiment, the IC₅₀ of the bifunctional compound is at most 5%, 4%, 3%,2%, 1%, 0.8%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% of the IC₅₀ of theTargeting Ligand. In one embodiment, the IC₅₀ of the bifunctionalcompound is at most 2%, 1%, 0.8%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% of theIC₅₀ of the Targeting Ligand. In one embodiment, the IC₅₀ of thebifunctional compound is at most 1%, 0.8%, 0.5%, 0.4%, 0.3%, 0.2%, or0.1% of the IC₅₀ of the Targeting Ligand. In one embodiment, thebifunctional compound inhibits the growth of cells (e.g., cancer cells)or decreases the viability of cells (e.g., cancer cells) at an E_(max)that is lower than the E_(max) of the Targeting Ligand (when theTargeting Ligand is administered alone (i.e., not bonded to a Linker anda Degron)) for inhibiting the growth or decreasing the viability of thecells. In one embodiment, the E_(max) of the bifunctional compound is atmost 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 8%, 5%, 4%, 3%, 2%, or1% of the E_(max) of the Targeting Ligand. In one embodiment, theE_(max) of the bifunctional compound is at most 50%, 40%, 30%, 20%, 10%,8%, 5%, 4%, 3%, 2%, or 1% of the E_(max) of the Targeting Ligand. In oneembodiment, the E_(max) of the bifunctional compound is at most 90%,80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10% of the E_(max) of theTargeting Ligand.

In some embodiments, the bifunctional compounds of Formula X, Formula Y,Formula Z, Formula A, Formula B, Formula C, or Formula D can modulateEGFR containing one or more mutations, such as those described herein,but not a wild-type EGFR. In some embodiments, the compounds of theapplication exhibit greater inhibition of EGFR containing one or moremutations as described herein relative to a wild-type EGFR. In certainembodiments, the bifunctional compounds of the application exhibit atleast 2-fold, 3-fold, 5-fold, 10-fold, 25-fold, 50-fold or 100-foldgreater inhibition of EGFR containing one or more mutations as describedherein relative to a wild-type EGFR. In various embodiments, thebifunctional compounds of the application exhibit up to 1000-foldgreater inhibition of EGFR containing one or more mutations as describedherein relative to a wild-type EGFR. In various embodiments, thebifunctional compounds of Formula X, Formula Y, Formula Z, Formula A,Formula B, Formula C, or Formula D exhibit up to 10000-fold greaterinhibition of EGFR having a combination of mutations described herein(e.g., L858R/T790M, L858R/T790M/I941R, L858R/T790M/C797S, Del/T790M,Del/T790M/C797S, and L858R/T790M) relative to a wild-type EGFR.

In some embodiments, the bifunctional compounds of Formula X, Formula Y,Formula Z, Formula A, Formula B, Formula C, or Formula D exhibit fromabout 2-fold to about 10-fold greater inhibition of EGFR containing oneor more mutations as described herein relative to a wild-type EGFR. Invarious embodiments, the bifunctional compounds of the applicationexhibit from about 10-fold to about 100-fold greater inhibition of EGFRcontaining one or more mutations as described herein relative to awild-type EGFR. In various embodiments, the bifunctional compounds ofthe application exhibit from about 100-fold to about 1000-fold greaterinhibition of EGFR containing one or more mutations as described hereinrelative to a wild-type EGFR. In various embodiments, the bifunctionalcompounds of the application exhibit from about 1000-fold to about10000-fold greater inhibition of EGFR containing one or more mutationsas described herein relative to a wild-type EGFR.

In certain embodiments, the bifunctional compounds of Formula X, FormulaY, Formula Z, Formula A, Formula B, Formula C, or Formula D exhibit atleast 2-fold greater inhibition of EGFR having a combination ofmutations selected from L858R/T790M, L858R/T790M/I941R,L858R/T790M/C797S, Del/T790M, Del/T790M/C797S, and L858R/T790M relativeto a wild-type EGFR. In certain embodiments, the bifunctional compoundsof the application exhibit at least 3-fold greater inhibition of EGFRhaving a combination of mutations selected from L858R/T790M,L858R/T790M/I941R, L858R/T790M/C797S, Del/T790M, Del/T790M/C797S, andL858R/T790M relative to a wild-type EGFR. In certain embodiments, thebifunctional compounds of the application exhibit at least 5-foldgreater inhibition of EGFR having a combination of mutations selectedfrom L858R/T790M, L858R/T790M/I941R, L858R/T790M/C797S, Del/T790M,Del/T790M/C797S, and L858R/T790M relative to a wild-type EGFR. Incertain embodiments, the bifunctional compounds of the applicationexhibit at least 10-fold greater inhibition of EGFR having a combinationof mutations selected from L858R/T790M, L858R/T790M/I941R,L858R/T790M/C797S, Del/T790M, Del/T790M/C797S, and L858R/T790M relativeto a wild-type EGFR. In certain embodiments, the bifunctional compoundsof the application exhibit at least 25-fold greater inhibition of EGFRhaving a combination of mutations selected from L858R/T790M,L858R/T790M/I941R, L858R/T790M/C797S, Del/T790M, Del/T790M/C797S, andL858R/T790M relative to a wild-type EGFR. In certain embodiments, thebifunctional compounds of the application exhibit at least 50-foldgreater inhibition of EGFR having a combination of mutations selectedfrom L L858R/T790M, L858R/T790M/I941R, L858R/T790M/C797S, Del/T790M,Del/T790M/C797S, and L858R/T790M relative to a wild-type EGFR. Incertain embodiments, the bifunctional compounds of the applicationexhibit at least 100-fold greater inhibition of EGFR having acombination of mutations selected from L858R/T790M, L858R/T790M/I941R,L858R/T790M/C797S, Del/T790M, Del/T790M/C797S, and L858R/T790M relativeto a wild-type EGFR.

In some embodiments, the inhibition of EGFR activity is measured byIC₅₀.

In some embodiments, the inhibition of EGFR activity is measured byEC₅₀.

In some embodiments, the bifunctional compounds of Formula X, Formula Y,Formula Z, Formula A, Formula B, Formula C, or Formula D bind to anallosteric site in EGFR. In some embodiments, the bifunctional compoundsof the application interact with at least one amino acid residue ofepidermal growth factor receptor (EGFR) selected from Lys745, Leu788,and Ala 743. In other embodiments, the bifunctional compounds of theapplication interact with at least one amino acid residue of epidermalgrowth factor receptor (EGFR) selected from Cys755, Leu777, Phe856, andAsp855. In other embodiments, the bifunctional compounds of theapplication interact with at least one amino acid residue of epidermalgrowth factor receptor (EGFR) selected from Met766, Ile759, Glu762, andAla763. In other embodiments, the bifunctional compounds of theapplication interact with at least one amino acid residue of epidermalgrowth factor receptor (EGFR) selected from Lys745, Leu788, and Ala 743,at least one amino acid residue of epidermal growth factor receptor(EGFR) selected from Cys755, Leu777, Phe856, and Asp855, and at leastone amino acid residue of epidermal growth factor receptor (EGFR)selected from Met766, Ile759, Glu762, and Ala763. In other embodiments,the bifunctional compounds of the application do not interact with theany of the amino acid residues of epidermal growth factor receptor(EGFR) selected from Met793, Gly796, and Cys797.

In some embodiments, the application provides a bifunctional compoundcomprising an allosteric kinase inhibitor, wherein the bifunctionalcompound is a more potent inhibitor of a drug-resistant EGFR mutantrelative to a wild type EGFR. For example, the bifunctional compound canbe at least about 2-fold, 3-fold, 5-fold, 10-fold, 25-fold, 50-fold orabout 100-fold more potent at inhibiting the kinase activity of thedrug-resistant EGFR mutant relative to a wild-type EGFR. In someembodiments, the drug-resistant EGFR mutant is resistant to one or moreknown EGFR inhibitors, including but not limited to gefitinib,erlotinib, lapatinib, WZ4002:

In some embodiments, the drug-resistant EGFR mutant comprises asensitizing mutation, such as Del and L858R.

In some embodiments, the application provides a bifunctional compoundcomprising an allosteric kinase inhibitor, wherein the compound inhibitskinase activity of a drug-resistant EGFR mutant harboring a sensitizingmutation (e.g., Del and L858R) and a drug-resistance mutation (e.g.,T790M, L718Q, C797S, and L844V) with less than a 10-fold difference inpotency (e.g., as measured by IC₅₀) relative to an EGFR mutant harboringthe sensitizing mutation but not the drug-resistance mutation. In someembodiments, the difference in potency is less than about 9-fold,8-fold, 7-fold, 6-fold, 5-fold, 4-fold, 3-fold, or 2-fold.

In some embodiments, the application provides a bifunctional compoundcomprising an allosteric kinase inhibitor, wherein the compound is morepotent than one or more known EGFR inhibitors, including, but notlimited to, gefitinib, erlotinib, lapatinib, WZ4002, HKI-272, CL-387785,and AZD9291, at inhibiting the activity of EGFR containing one or moremutations as described herein, such as T790M, L718Q, L844V, L858R,C797S, and Del. For example, the compound can be at least about 2-fold,3-fold, 5-fold, 10-fold, 25-fold, 50-fold or about 100-fold more potent(e.g., as measured by IC₅₀) than gefitinib, erlotinib, lapatinib,WZ4002, HKI-272, CL-387785, and AZD9291 at inhibiting the activity ofthe EGFR containing one or more mutations as described herein.

In some embodiments, the application provides a bifunctional compoundcomprising an allosteric kinase inhibitor, wherein the bifunctionalcompound is less potent than one or more known EGFR inhibitors,including but not limited to gefitinib, erlotinib, lapatinib, WZ4002,HKI-272, CL-387785, and AZD9291, at inhibiting the activity of awild-type EGFR. For example, the bifunctional compound can be at leastabout 2-fold, 3-fold, 5-fold, 10-fold, 25-fold, 50-fold or about100-fold less potent (e.g., as measured by IC₅₀) than gefitinib,erlotinib, lapatinib, WZ4002, HKI-272, CL-387785, and AZD9291, atinhibiting the activity of a wild-type EGFR.

Potency of the inhibitor can be determined by EC₅₀ value. A compoundwith a lower EC₅₀ value, as determined under substantially similarconditions, is a more potent inhibitor relative to a compound with ahigher EC₅₀ value. In some embodiments, the substantially similarconditions comprise determining an EGFR-dependent phosphorylation levelor determining cell proliferation or cell death, in vitro or in vivo(e.g., in 3T3 cells expressing a wild type EGFR, a mutant EGFR, or afragment of any thereof).

Potency of the inhibitor can also be determined by IC₅₀ value. Acompound with a lower IC₅₀ value, as determined under substantiallysimilar conditions, is a more potent inhibitor relative to a compoundwith a higher IC₅₀ value. In some embodiments, the substantially similarconditions comprise determining an EGFR-dependent phosphorylation levelor determining cell proliferation or cell death, in vitro or in vivo(e.g., in 3T3 cells expressing a wild type EGFR, a mutant EGFR, or afragment of any thereof).

An EGFR sensitizing mutation comprises without limitation L858R, G719S,G719C, G719A, L861Q, a deletion in exon 19 and/or an insertion in exon20. A drug-resistant EGFR mutant can have without limitation a drugresistance mutation comprising T790M, T854A, L718Q, C797S, or D761Y.

The selectivity between wild-type EGFR and EGFR containing one or moremutations as described herein can also be measured using cellularproliferation assays where cell proliferation is dependent on kinaseactivity. For example, murine Ba/F3 cells transfected with a suitableversion of wild-type EGFR (such as VIII; containing a WT EGFR kinasedomain), or Ba/F3 cells transfected with L858R/T790M, Del/T790M/L718Q,L858R/T790M/L718Q, L858R/T790M/C797S, Del/T790M/C797S,L858R/T790M/I941R, or Exon 19 deletion/T790M can be used. Proliferationassays are performed at a range of inhibitor concentrations (10 μM, 3μM, 1.1 μM, 330 nM, 110 nM, 33 nM, 11 nM, 3 nM, I nM) and an EC₅₀ iscalculated.

An alternative method to measure effects on EGFR activity is to assayEGFR phosphorylation. Wild type or mutant (L858R/T790M, Del/T790M,Del/T790M/L718Q, L858R/T790M/C797S, Del/T790M/C797S, L858R/T790M/I941R,or L858R/T790M/L718Q) EGFR can be transfected into NIH-3T3 cells (whichdo not normally express endogenous EGFR) and the ability of theinhibitor (using concentrations as above) to inhibit EGFRphosphorylation can be assayed. Cells are exposed to increasingconcentrations of inhibitor for 6 hours and stimulated with EGF for 10minutes. The effects on EGFR phosphorylation are assayed by WesternBlotting using phospho-specific (Y1068) EGFR antibodies.

In another aspect, the present application provides a bifunctionalcompound that binds to an allosteric site in EGFR, wherein the compoundexhibits greater than 2-fold, 3-fold, 5-fold, 10-fold, 25-fold, 50-fold,100-fold, or 1000-fold inhibition of EGFR containing one or moremutations as described herein (e.g., L858R/T790M, Del/T790M,Del/T790M/L718Q, L858R/T790M/C797S, Del/T790M/C797S, L858R/T790M/I941R,or L858R/T790M/L718Q) relative to a wild-type EGFR.

In one embodiment, the bifunctional compounds of Formula X, Formula Y,Formula Z, Formula A, Formula B, Formula C, or Formula D are useful asanticancer agents, and thus may be useful in the treatment of cancer, byeffecting tumor cell death or inhibiting the growth of tumor cells. Incertain exemplary embodiments, the disclosed anticancer agents areuseful in the treatment of cancers and other proliferative disorders,including, but not limited to breast cancer, cervical cancer, colon andrectal cancer, leukemia, lung cancer (e.g., non-small cell lung cancer),melanoma, multiple myeloma, non-Hodgkin's lymphoma, ovarian cancer,pancreatic cancer, prostate cancer, gastric cancer, leukemias (e.g.,myeloid, lymphocytic, myelocytic and lymphoblastic leukemias), malignantmelanomas, and T-cell lymphoma.

Definitions

Listed below are definitions of various terms used in this application.These definitions apply to the terms as they are used throughout thisspecification and claims, unless otherwise limited in specificinstances, either individually or as part of a larger group.

The term “alkyl,” as used herein, refers to saturated, straight orbranched-chain hydrocarbon radicals containing, in certain embodiments,between one and six carbon atoms. Examples of C₁-C₆ alkyl radicalsinclude, but are not limited to, methyl, ethyl, propyl, isopropyl,n-butyl, tert-butyl, neopentyl, and n-hexyl radicals.

The term “alkenyl,” as used herein, denotes a monovalent group derivedfrom a hydrocarbon moiety containing, in certain embodiments, from twoto six carbon atoms having at least one carbon-carbon double bond. Thedouble bond may or may not be the point of attachment to another group.Alkenyl groups include, but are not limited to, for example, ethenyl,propenyl, butenyl, 1-methyl-2-buten-1-yl and the like.

The term “alkoxy” refers to an —O-alkyl radical.

The terms “hal,” “halo,” and “halogen,” as used herein, refer to an atomselected from fluorine, chlorine, bromine and iodine.

The term “aryl,” as used herein, refers to a mono- or poly-cycliccarbocyclic ring system having one or more aromatic rings, fused ornon-fused, including, but not limited to, phenyl, naphthyl,tetrahydronaphthyl, indanyl, indenyl and the like.

The term “aralkyl,” as used herein, refers to an alkyl residue attachedto an aryl ring. Examples include, but are not limited to, benzyl,phenethyl and the like.

The term “cycloalkyl,” as used herein, denotes a monovalent groupderived from a monocyclic or polycyclic saturated or partiallyunsaturated carbocyclic ring compound. Examples of C₃-C₈ cycloalkylinclude, but not limited to, cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, cyclopentyl and cyclooctyl; and examples ofC₃-C₁₂-cycloalkyl include, but not limited to, cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, bicyclo [2.2.1] heptyl, and bicyclo [2.2.2]octyl. Also contemplated is a monovalent group derived from a monocyclicor polycyclic carbocyclic ring compound having at least onecarbon-carbon double bond by the removal of a single hydrogen atom.Examples of such groups include, but are not limited to, cyclopropenyl,cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl,and the like.

The term “heteroaryl,” as used herein, refers to a mono- or poly-cyclic(e.g., bi-, or tri-cyclic or more) fused or non-fused, radical or ringsystem having at least one aromatic ring, having from five to ten ringatoms of which one ring atoms is selected from S, O, and N; zero, one,or two ring atoms are additional heteroatoms independently selected fromS, O, and N; and the remaining ring atoms are carbon. Heteroarylincludes, but is not limited to, pyridinyl, pyrazinyl, pyrimidinyl,pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl,thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl,isoquinolinyl, benzimidazolyl, benzooxazolyl, quinoxalinyl, and thelike.

The term “heteroaralkyl,” as used herein, refers to an alkyl residueattached to a heteroaryl ring. Examples include, but are not limited to,pyridinylmethyl, pyrimidinylethyl and the like.

The term “heterocyclyl,” or “heterocycloalkyl,” as used herein, refersto a non-aromatic 3-4-, 5-, 6- or 7-membered ring or a bi- or tri-cyclicgroup fused of non-fused system, where (i) each ring contains betweenone and three heteroatoms independently selected from oxygen, sulfur andnitrogen, (ii) each 5-membered ring has 0 to 1 double bonds and each6-membered ring has 0 to 2 double bonds, (iii) the nitrogen and sulfurheteroatoms may optionally be oxidized, and (iv) the nitrogen heteroatommay optionally be quaternized. Representative heterocycloalkyl groupsinclude, but are not limited to, [1,3]dioxolane, pyrrolidinyl,pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl,piperazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl,isothiazolidinyl, and tetrahydrofuryl.

The term “alkylamino” refers to a group having the structure —NH(C₁-C₁₂alkyl), e.g., —NH(C₁-C₆ alkyl), where C₁-C₁₂ alkyl is as previouslydefined.

The term “dialkylamino” refers to a group having the structure —N(C₁-C₁₂alkyl)₂, e.g., —NH(C₁-C₆ alkyl), where C₁-C₁₂ alkyl is as previouslydefined.

The term “acyl” includes residues derived from acids, including but notlimited to carboxylic acids, carbamic acids, carbonic acids, sulfonicacids, and phosphorous acids. Examples include aliphatic carbonyls,aromatic carbonyls, aliphatic sulfonyls, aromatic sulfinyls, aliphaticsulfinyls, aromatic phosphates and aliphatic phosphates. Examples ofaliphatic carbonyls include, but are not limited to, acetyl, propionyl,2-fluoroacetyl, butyryl, 2-hydroxy acetyl, and the like.

In accordance with the application, any of the aryls, substituted aryls,heteroaryls and substituted heteroaryls described herein, can be anyaromatic group. Aromatic groups can be substituted or unsubstituted.

The terms “hal,” “halo,” and “halogen,” as used herein, refer to an atomselected from fluorine, chlorine, bromine and iodine.

As described herein, compounds of the application may optionally besubstituted with one or more substituents, such as are illustratedgenerally above, or as exemplified by particular classes, subclasses,and species of the application. It will be appreciated that the phrase“optionally substituted” is used interchangeably with the phrase“substituted or unsubstituted.” In general, the term “substituted”,whether preceded by the term “optionally” or not, refers to thereplacement of hydrogen radicals in a given structure with the radicalof a specified substituent. Unless otherwise indicated, an optionallysubstituted group may have a substituent at each substitutable positionof the group, and when more than one position in any given structure maybe substituted with more than one substituent selected from a specifiedgroup, the substituent may be either the same or different at everyposition. The terms “optionally substituted”, “optionally substitutedalkyl,” “optionally substituted “optionally substituted alkenyl,”“optionally substituted alkynyl”, “optionally substituted cycloalkyl,”“optionally substituted cycloalkenyl,” “optionally substituted aryl”,“optionally substituted heteroaryl,” “optionally substituted aralkyl”,“optionally substituted heteroaralkyl,” “optionally substitutedheterocycloalkyl,” and any other optionally substituted group as usedherein, refer to groups that are substituted or unsubstituted byindependent replacement of one, two, or three or more of the hydrogenatoms thereon with substituents including, but not limited to:

—F, —Cl, —Br, —I, —OH, protected hydroxy, —NO₂, —CN, —NH₂, protectedamino, —NH—C₁-C₁₂-alkyl, —NH—C₂-C₁₂-alkenyl, —NH—C₂-C₁₂-alkenyl,—NH—C₃-C₁₂-cycloalkyl, —NH-aryl, —NH-heteroaryl, —NH-heterocycloalkyl,-dialkylamino, -diarylamino, -diheteroarylamino, —O—C₁-C₁₂-alkyl,—O—C₂-C₁₂-alkenyl, —O—C₂-C₁₂-alkenyl, —O—C₃-C₁₂-cycloalkyl, —O-aryl,—O-heteroaryl, —O-heterocycloalkyl, —C(O)—C₁-C₁₂-alkyl,—C(O)—C₂-C₁₂-alkenyl, —C(O)—C₂-C₁₂-alkenyl, —C(O)—C₃-C₁₂-cycloalkyl,—C(O)-aryl, —C(O)-heteroaryl, —C(O)-heterocycloalkyl, —CONH₂,—CONH—C₁-C₁₂-alkyl, —CONH—C₂-C₁₂-alkenyl, —CONH—C₂-C₁₂-alkenyl,—CONH—C₃-C₁₂-cycloalkyl, —CONH-aryl, —CONH-heteroaryl,—CONH-heterocycloalkyl, —OCO₂—C₁-C₁₂-alkyl, —OCO₂—C₂-C₁₂-alkenyl,—OCO₂—C₂-C₁₂-alkenyl, —OCO₂—C₃-C₁₂-cycloalkyl, —OCO₂-aryl,—OCO₂-heteroaryl, —OCO₂-heterocycloalkyl, —OCONH₂, —OCONH—C₁-C₁₂-alkyl,—OCONH—C₂-C₁₂-alkenyl, —OCONH—C₂-C₁₂-alkenyl, —OCONH—C₃-C₁₂-cycloalkyl,—OCONH-aryl, —OCONH-heteroaryl, —OCONH-heterocycloalkyl,—NHC(O)—C₁-C₁₂-alkyl, —NHC(O)—C₂-C₁₂-alkenyl, —NHC(O)—C₂-C₁₂-alkenyl,—NHC(O)—C₃-C₁₂-cycloalkyl, —NHC(O)-aryl, —NHC(O)-heteroaryl,—NHC(O)-heterocycloalkyl, —NHCO₂—C₁-C₁₂-alkyl, —NHCO₂—C₂-C₁₂-alkenyl,—NHCO₂—C₂-C₁₂-alkenyl, —NHCO₂—C₃-C₁₂-cycloalkyl, —NHCO₂-aryl,—NHCO₂-heteroaryl, —NHCO₂— heterocycloalkyl, NHC(O)NH₂,—NHC(O)NH—C₁-C₁₂-alkyl, —NHC(O)NH—C₂-C₁₂-alkenyl,—NHC(O)NH—C₂-C₁₂-alkenyl, —NHC(O)NH—C₃-C₁₂-cycloalkyl, —NHC(O)NH-aryl,—NHC(O)NH-heteroaryl, NHC(O)NH-heterocycloalkyl, —NHC(S)NH₂,—NHC(S)NH—C₁-C₁₂-alkyl, —NHC(S)NH—C₂-C₁₂-alkenyl,—NHC(S)NH—C₂-C₁₂-alkenyl, —NHC(S)NH—C₃-C₁₂-cycloalkyl, —NHC(S)NH-aryl,—NHC(S)NH-heteroaryl, —NHC(S)NH-heterocycloalkyl, —NHC(NH)NH₂,—NHC(NH)NH—C₁-C₁₂-alkyl, —NHC(NH)NH—C₂-C₁₂-alkenyl,—NHC(NH)NH—C₂-C₁₂-alkenyl, —NHC(NH)NH—C₃-C₁₂-cycloalkyl,—NHC(NH)NH-aryl, —NHC(NH)NH-heteroaryl, —NHC(NH)NHheterocycloalkyl,—NHC(NH)—C₁-C₁₂-alkyl, —NHC(NH)—C₂-C₁₂-alkenyl, —NHC(NH)—C₂-C₁₂-alkenyl,—NHC(NH)—C₃-C₁₂-cycloalkyl, —NHC(NH)-aryl, —NHC(NH)-heteroaryl,—NHC(NH)-heterocycloalkyl, —C(NH)NH—C₁-C₁₂-alkyl,—C(NH)NH—C₂-C₁₂-alkenyl, —C(NH)NH—C₂-C₁₂-alkenyl,C(NH)NH—C₃-C₁₂-cycloalkyl, —C(NH)NH-aryl, —C(NH)NH-heteroaryl,—C(NH)NHheterocycloalkyl, —S(O)—C₁-C₁₂-alkyl, —S(O)—C₂-C₁₂-alkenyl,—S(O)—C₂-C₁₂-alkenyl, —S(O)—C₃-C₁₂-cycloalkyl, —S(O)-aryl,—S(O)-heteroaryl, —S(O)-heterocycloalkyl —SO₂NH₂, —SO₂NH—C₁-C₁₂-alkyl,—SO₂NH—C₂-C₁₂-alkenyl, —SO₂NH—C₂-C₁₂-alkenyl, —SO₂NH—C₃-C₁₂-cycloalkyl,—SO₂NH-aryl, —SO₂NH-heteroaryl, —SO₂NH-heterocycloalkyl,—NHSO₂—C₁-C₁₂-alkyl, —NHSO₂—C₂-C₁₂-alkenyl, —NHSO₂—C₂-C₁₂-alkenyl,—NHSO₂—C₃-C₁₂-cycloalkyl, —NHSO₂-aryl, —NHSO₂-heteroaryl,—NHSO₂-heterocycloalkyl, —CH₂NH₂, —CH₂SO₂CH₃, -aryl, -arylalkyl,-heteroaryl, -heteroarylalkyl, -heterocycloalkyl, —C₃-C₁₂-cycloalkyl,polyalkoxyalkyl, polyalkoxy, -methoxymethoxy, -methoxyethoxy, —SH,—S—C₁-C₁₂-alkyl, —S—C₂-C₁₂-alkenyl, —S—C₂-C₁₂-alkenyl,—S—C₃-C₁₂-cycloalkyl, —S-aryl, —S-heteroaryl, —S-heterocycloalkyl, ormethylthiomethyl.

It is understood that the aryls, heteroaryls, alkyls, and the like canbe substituted.

The term “cancer” includes, but is not limited to, the followingcancers: epidermoid Oral: buccal cavity, lip, tongue, mouth, pharynx;Cardiac: sarcoma (angiosarcoma, fibrosarcoma, rhabdomyosarcoma,liposarcoma), myxoma, rhabdomyoma, fibroma, lipoma, and teratoma; Lung:bronchogenic carcinoma (squamous cell or epidermoid, undifferentiatedsmall cell, undifferentiated large cell, adenocarcinoma), alveolar(bronchiolar) carcinoma, bronchial adenoma, sarcoma, lymphoma,chondromatous hamartoma, mesothelioma; Gastrointestinal: esophagus(squamous cell carcinoma, larynx, adenocarcinoma, leiomyosarcoma,lymphoma), stomach (carcinoma, lymphoma, leiomyosarcoma), pancreas(ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoidtumors, vipoma), small bowel or small intestines (adenocarcinoma,lymphoma, carcinoid tumors, Karposi's sarcoma, leiomyoma, hemangioma,lipoma, neurofibroma, fibroma), large bowel or large intestines(adenocarcinoma, tubular adenoma, villous adenoma, hamartoma,leiomyoma), colon, colon-rectum, colorectal, rectum; Genitourinarytract: kidney (adenocarcinoma, Wilm's tumor (nephroblastoma), lymphoma,leukemia), bladder and urethra (squamous cell carcinoma, transitionalcell carcinoma, adenocarcinoma), prostate (adenocarcinoma, sarcoma),testis (seminoma, teratoma, embryonal carcinoma, teratocarcinoma,choriocarcinoma, sarcoma, interstitial cell carcinoma, fibroma,fibroadenoma, adenomatoid tumors, lipoma); Liver: hepatoma(hepatocellular carcinoma), cholangiocarcinoma, hepatoblastoma,angiosarcoma, hepatocellular adenoma, hemangioma, biliary passages;Bone: osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant fibroushistiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma(reticulum cell sarcoma), multiple myeloma, malignant giant cell tumorchordoma, osteochronfroma (osteocartilaginous exostoses), benignchondroma, chondroblastoma, chondromyxofibroma, osteoid osteoma andgiant cell tumors; Nervous system: skull (osteoma, hemangioma,granuloma, xanthoma, osteitis deformans), meninges (meningioma,meningiosarcoma, gliomatosis), brain (astrocytoma, medulloblastoma,glioma, ependymoma, germinoma (pinealoma), glioblastoma multiform,oligodendroglioma, schwannoma, retinoblastoma, congenital tumors),spinal cord neurofibroma, meningioma, glioma, sarcoma); Gynecological:uterus (endometrial carcinoma), cervix (cervical carcinoma, pre-tumorcervical dysplasia), ovaries (ovarian carcinoma (serouscystadenocarcinoma, mucinous cystadenocarcinoma, unclassifiedcarcinoma), granulosa-thecal cell tumors, Sertoli-Leydig cell tumors,dysgerminoma, malignant teratoma), vulva (squamous cell carcinoma,intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma),vagina (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma(embryonal rhabdomyosarcoma), fallopian tubes (carcinoma), breast;Hematologic: blood (myeloid leukemia (acute and chronic), acutelymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferativediseases, multiple myeloma, myelodysplastic syndrome), Hodgkin'sdisease, non-Hodgkin's lymphoma (malignant lymphoma) hairy cell;lymphoid disorders; Skin: malignant melanoma, basal cell carcinoma,squamous cell carcinoma, Karposi's sarcoma, keratoacanthoma, molesdysplastic nevi, lipoma, angioma, dermatofibroma, keloids, psoriasis,Thyroid gland: papillary thyroid carcinoma, follicular thyroidcarcinoma; medullary thyroid carcinoma, undifferentiated thyroid cancer,multiple endocrine neoplasia type 2A, multiple endocrine neoplasia type2B, familial medullary thyroid cancer, pheochromocytoma, paraganglioma;and Adrenal glands: neuroblastoma. Thus, the term “cancerous cell” asprovided herein, includes a cell afflicted by any one of theabove-identified conditions.

The term “EGFR” herein refers to epidermal growth factor receptorkinase.

The term “HER” or “Her”, herein refers to human epidermal growth factorreceptor kinase.

The term “subject” as used herein refers to a mammal. A subjecttherefore refers to, for example, dogs, cats, horses, cows, pigs, guineapigs, and the like. Preferably the subject is a human. When the subjectis a human, the subject may be referred to herein as a patient.

“Treat”, “treating” and “treatment” refer to a method of alleviating orabating a disease and/or its attendant symptoms.

As used herein, “preventing” or “prevent” describes reducing oreliminating the onset of the symptoms or complications of the disease,condition or disorder.

As used herein, the term “allosteric site” refers to a site on EGFRother than the ATP binding site, such as that characterized in a crystalstructure of EGFR. An “allosteric site” can be a site that is close tothe ATP binding site, such as that characterized in a crystal structureof EGFR. For example, one allosteric site includes one or more of thefollowing amino acid residues of epidermal growth factor receptor(EGFR): Lys745, Leu788, Ala 743, Cys755, Leu777, Phe856, Asp855, Met766,Ile759, Glu762, and/or Ala763.

As used herein, the term “agent that prevents EGFR dimer formation”refers to an agent that prevents dimer formation in which the C-lobe ofthe “activator” subunit impinges on the N-lobe of the “receiver”subunit. Examples of agents that prevent EGFR dimer formation include,but are not limited to, cetuximab, cobimetinib, trastuzumab,panitumumab, and Mig6.

As used herein the term “GDC0973” or “Cobimetinib” refers to a compoundhaving the chemical structure:

As used herein, “TL compound 1,” and “TL compound 2,” refers tocompounds having the chemical structures.

The term “targeted protein(s)” is used interchangeably with “targetprotein(s)”, unless the context clearly dictates otherwise. In oneembodiment, a “targeted protein” is EGFR.

The term “subject” as used herein refers to a mammal. A subjecttherefore refers to, for example, dogs, cats, horses, cows, pigs, guineapigs, and the like. Preferably the subject is a human. When the subjectis a human, the subject may be referred to herein as a patient.

The terms “disease(s)”, “disorder(s)”, and “condition(s)” are usedinterchangeably, unless the context clearly dictates otherwise.

The term “therapeutically effective amount” of a bifunctional compoundor pharmaceutical composition of the application, as used herein, meansa sufficient amount of the bifunctional compound or pharmaceuticalcomposition so as to decrease the symptoms of a disorder in a subject.As is well understood in the medical arts a therapeutically effectiveamount of a bifunctional compound or pharmaceutical composition of thisapplication will be at a reasonable benefit/risk ratio applicable to anymedical treatment. It will be understood, however, that the total dailyusage of the compounds and compositions of the present application willbe decided by the attending physician within the scope of sound medicaljudgment. The specific inhibitory dose for any particular patient willdepend upon a variety of factors including the disorder being treatedand the severity of the disorder; the activity of the specific compoundemployed; the specific composition employed; the age, body weight,general health, sex and diet of the patient; the time of administration,route of administration, and rate of excretion of the specific compoundemployed; the duration of the treatment; drugs used in combination orcoincidental with the specific compound employed; and like factors wellknown in the medical arts.

As used herein, the term “pharmaceutically acceptable salt” refers tothose salts of the compounds formed by the process of the presentapplication which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of humans and lower animalswithout undue toxicity, irritation, allergic response and the like, andare commensurate with a reasonable benefit/risk ratio. Pharmaceuticallyacceptable salts are well known in the art. For example, S. M. Berge, etal. describes pharmaceutically acceptable salts in detail in J.Pharmaceutical Sciences, 66: 1-19 (1977). The salts can be prepared insitu during the final isolation and purification of the compounds of theapplication, or separately by reacting the free base or acid functionwith a suitable acid or base.

Examples of pharmaceutically acceptable salts include, but are notlimited to, nontoxic acid addition salts: salts formed with inorganicacids such as hydrochloric acid, hydrobromic acid, phosphoric acid,sulfuric acid and perchloric acid, or with organic acids such as aceticacid, maleic acid, tartaric acid, citric acid, succinic acid or malonicacid. Other pharmaceutically acceptable salts include, but are notlimited to, adipate, alginate, ascorbate, aspartate, benzenesulfonate,benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate,citrate, cyclopentanepropionate, digluconate, dodecylsulfate,ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate,gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide,2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, laurylsulfate, malate, maleate, malonate, methanesulfonate,2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate,pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate,pivalate, propionate, stearate, succinate, sulfate, tartrate,thiocyanate, /7-toluenesulfonate, undecanoate, valerate salts, and thelike. Representative alkali or alkaline earth metal salts includesodium, lithium, potassium, calcium, magnesium, and the like. Furtherpharmaceutically acceptable salts include, when appropriate, nontoxicammonium, quaternary ammonium, and amine cations formed usingcounterions such as halide, hydroxide, carboxylate, sulfate, phosphate,nitrate, alkyl having from 1 to 6 carbon atoms, sulfonate and arylsulfonate.

As used herein, the term “pharmaceutically acceptable ester” refers toesters of the bifunctional compounds formed by the process of thepresent application which hydrolyze in vivo and include those that breakdown readily in the human body to leave the parent compound or a saltthereof. Suitable ester groups include, for example, those derived frompharmaceutically acceptable aliphatic carboxylic acids, particularlyalkanoic, alkenoic, cycloalkanoic and alkanedioic acids, in which eachalkyl or alkenyl moiety advantageously has not more than 6 carbon atoms.Examples of particular esters include, but are not limited to, formates,acetates, propionates, butyrates, acrylates and ethylsuccinates.

The term “pharmaceutically acceptable prodrugs” as used herein refers tothose prodrugs of the bifunctional compounds formed by the process ofthe present application which are, within the scope of sound medicaljudgment, suitable for use in contact with the tissues of humans andlower animals with undue toxicity, irritation, allergic response, andthe like, commensurate with a reasonable benefit/risk ratio, andeffective for their intended use, as well as the zwitterionic forms,where possible, of the compounds of the present application. “Prodrug”,as used herein means a compound which is convertible in vivo bymetabolic means (e.g., by hydrolysis) to afford any compound delineatedby the formulae of the instant application. Various forms of prodrugsare known in the art, for example, as discussed in Bundgaard, (ed.),Design of Prodrugs, Elsevier (1985); Widder, et al. (ed.), Methods inEnzymology, vol. 4, Academic Press (1985); Krogsgaard-Larsen, et al.,(ed). “Design and Application of Prodrugs, Textbook of Drug Design andDevelopment, Chapter 5, 113-191 (1991); Bundgaard, et al., Journal ofDrug Deliver Reviews, 8:1-38(1992); Bundgaard, J. of PharmaceuticalSciences, 77:285 et seq. (1988); Higuchi and Stella (eds.) Prodrugs asNovel Drug Delivery Systems, American Chemical Society (1975); andBernard Testa & Joachim Mayer, “Hydrolysis In Drug And ProdrugMetabolism: Chemistry, Biochemistry And Enzymology,” John Wiley andSons, Ltd. (2002).

This application also encompasses pharmaceutical compositionscontaining, and methods of treating disorders through administering,pharmaceutically acceptable prodrugs of bifunctional compounds of theapplication. For example, compounds of the application having freeamino, amido, hydroxy or carboxylic groups can be converted intoprodrugs. Prodrugs include compounds wherein an amino acid residue, or apolypeptide chain of two or more (e.g., two, three or four) amino acidresidues is covalently joined through an amide or ester bond to a freeamino, hydroxy or carboxylic acid group of compounds of the application.The amino acid residues include but are not limited to the 20 naturallyoccurring amino acids commonly designated by three letter symbols andalso includes 4-hydroxyproline, hydroxylysine, demosine, isodemosine,3-methylhistidine, norvalin, beta-alanine, gamma-aminobutyric acid,citrulline, homocysteine, homoserine, ornithine and methionine sulfone.Additional types of prodrugs are also encompassed. For instance, freecarboxyl groups can be derivatized as amides or alkyl esters. Freehydroxy groups may be derivatized using groups including but not limitedto hemisuccinates, phosphate esters, dimethylaminoacetates, andphosphoryloxymethyloxy carbonyls, as outlined in Advanced Drug DeliveryReviews, 1996, 19, 1 15. Carbamate prodrugs of hydroxy and amino groupsare also included, as are carbonate prodrugs, sulfonate esters andsulfate esters of hydroxy groups. Derivatization of hydroxy groups as(acyloxy)methyl and (acyloxy)ethyl ethers wherein the acyl group may bean alkyl ester, optionally substituted with groups including but notlimited to ether, amine and carboxylic acid functionalities, or wherethe acyl group is an amino acid ester as described above, are alsoencompassed. Prodrugs of this type are described in J. Med. Chem. 1996,39, 10. Free amines can also be derivatized as amides, sulfonamides orphosphonamides. All of these prodrug moieties may incorporate groupsincluding but not limited to ether, amine and carboxylic acidfunctionalities.

The application also provides for a pharmaceutical compositioncomprising a bifunctional compound disclosed herein, or apharmaceutically acceptable ester, salt, or prodrug thereof, togetherwith a pharmaceutically acceptable carrier.

In another aspect, the application provides a kit comprising abifunctional compound capable of inhibiting EGFR activity selected fromone or more compounds disclosed herein, or a pharmaceutically acceptablesalt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof,optionally in combination with a second agent wherein said second agentprevents EGFR dimer formation and instructions for use in treatingcancer.

In another aspect, the application provides a method of synthesizing abifunctional compound disclosed herein.

The synthesis of the bifunctional compounds of the application can befound herein and in the Examples below.

Other embodiments are a method of making a bifunctional compound of anyof the formulae herein using any one, or combination of, reactionsdelineated herein. The method can include the use of one or moreintermediates or chemical reagents delineated herein.

Another aspect is an isotopically labeled bifunctional compound of anyof the formulae delineated herein. Such compounds have one or moreisotope atoms which may or may not be radioactive (e.g., ³H, ²H, ¹⁴C,¹³C, ¹⁸F, ³⁵S, ³²P, ¹²⁵I, and ¹³¹I) introduced into the bifunctionalcompound. Such compounds are useful for drug metabolism studies anddiagnostics, as well as therapeutic applications.

A bifunctional compound of the application can be prepared as apharmaceutically acceptable acid addition salt by reacting the free baseform of the compound with a pharmaceutically acceptable inorganic ororganic acid. Alternatively, a pharmaceutically acceptable base additionsalt of a bifunctional compound of the application can be prepared byreacting the free acid form of the bifunctional compound with apharmaceutically acceptable inorganic or organic base.

Alternatively, the salt forms of the bifunctional compounds of theapplication can be prepared using salts of the starting materials orintermediates.

The free acid or free base forms of the bifunctional compounds of theapplication can be prepared from the corresponding base addition salt oracid addition salt from, respectively. For example, a bifunctionalcompound of the application in an acid addition salt form can beconverted to the corresponding free base by treating with a suitablebase (e.g., ammonium hydroxide solution, sodium hydroxide, and thelike). A bifunctional compound of the application in a base additionsalt form can be converted to the corresponding free acid by treatingwith a suitable acid (e.g., hydrochloric acid, etc.).

Prodrugs of the bifunctional compounds of the application can beprepared by methods known to those of ordinary skill in the art (e.g.,for further details see Saulnier et al., (1994), Bioorganic andMedicinal Chemistry Letters, Vol. 4, p. 1985). For example, appropriateprodrugs can be prepared by reacting a non-derivatized bifunctionalcompound of the application with a suitable carbamylating agent (e.g.,1,1-acyloxyalkylcarbanochloridate, para-nitrophenyl carbonate, or thelike).

Protected derivatives of the bifunctional compounds of the applicationcan be made by means known to those of ordinary skill in the art. Adetailed description of techniques applicable to the creation ofprotecting groups and their removal can be found in T. W. Greene,“Protecting Groups in Organic Chemistry”, 3rd edition, John Wiley andSons, Inc., 1999.

Compounds of the present application can be conveniently prepared, orformed during the process of the application, as solvates (e.g.,hydrates). Hydrates of bifunctional compounds of the present applicationcan be conveniently prepared by recrystallization from anaqueous/organic solvent mixture, using organic solvents such as dioxin,tetrahydrofuran or methanol.

Acids and bases useful in the methods herein are known in the art. Acidcatalysts are any acidic chemical, which can be inorganic (e.g.,hydrochloric, sulfuric, nitric acids, aluminum trichloride) or organic(e.g., camphorsulfonic acid, p-toluenesulfonic acid, acetic acid,ytterbium triflate) in nature. Acids are useful in either catalytic orstoichiometric amounts to facilitate chemical reactions. Bases are anybasic chemical, which can be inorganic (e.g., sodium bicarbonate,potassium hydroxide) or organic (e.g., triethylamine, pyridine) innature. Bases are useful in either catalytic or stoichiometric amountsto facilitate chemical reactions.

Combinations of substituents and variables envisioned by thisapplication are only those that result in the formation of stablecompounds. The term “stable”, as used herein, refers to compounds whichpossess stability sufficient to allow manufacture and which maintainsthe integrity of the compound for a sufficient period of time to beuseful for the purposes detailed herein (e.g., therapeutic orprophylactic administration to a subject).

When any variable (e.g., R₁₄) occurs more than one time in anyconstituent or formula for a compound, its definition at each occurrenceis independent of its definition at every other occurrence. Thus, forexample, if a group is shown to be substituted with one or more R₁₄moieties, then R₁₄ at each occurrence is selected independently from thedefinition of R₁₄. Also, combinations of substituents and/or variablesare permissible, but only if such combinations result in stablecompounds within a designated atom's normal valency.

In addition, some of the compounds of this application have one or moredouble bonds, or one or more asymmetric centers. Such compounds canoccur as racemates, racemic mixtures, single enantiomers, individualdiastereomers, diastereomeric mixtures, and cis- or trans- or E- orZ-double isomeric forms, and other stereoisomeric forms that may bedefined, in terms of absolute stereochemistry, as (R)- or (S)-, or as(D)- or (L)- for amino acids. When the compounds described hereincontain olefinic double bonds or other centers of geometric asymmetry,and unless specified otherwise, it is intended that the compoundsinclude both E and Z geometric isomers. The configuration of anycarbon-carbon double bond appearing herein is selected for convenienceonly and is not intended to designate a particular configuration unlessthe text so states; thus a carbon-carbon double bond depictedarbitrarily herein as trans may be cis, trans, or a mixture of the twoin any proportion. All such isomeric forms of such compounds areexpressly included in the present application.

Optical isomers may be prepared from their respective optically activeprecursors by the procedures described herein, or by resolving theracemic mixtures. The resolution can be carried out in the presence of aresolving agent, by chromatography or by repeated crystallization or bysome combination of these techniques which are known to those skilled inthe art. Further details regarding resolutions can be found in Jacques,et al., Enantiomers, Racemates, and Resolutions (John Wiley & Sons,1981).

“Isomerism” means compounds that have identical molecular formulae butdiffer in the sequence of bonding of their atoms or in the arrangementof their atoms in space. Isomers that differ in the arrangement of theiratoms in space are termed “stereoisomers”. Stereoisomers that are notmirror images of one another are termed “diastereoisomers”, andstereoisomers that are non-superimposable mirror images of each otherare termed “enantiomers” or sometimes optical isomers. A mixturecontaining equal amounts of individual enantiomeric forms of oppositechirality is termed a “racemic mixture”.

A carbon atom bonded to four non-identical substituents is termed a“chiral center”.

“Chiral isomer” means a compound with at least one chiral center.Compounds with more than one chiral center may exist either as anindividual diastereomer or as a mixture of diastereomers, termed“diastereomeric mixture”. When one chiral center is present, astereoisomer may be characterized by the absolute configuration (R or S)of that chiral center. Absolute configuration refers to the arrangementin space of the substituents attached to the chiral center. Thesubstituents attached to the chiral center under consideration areranked in accordance with the Sequence Rule of Cahn, Ingold and Prelog.(Cahn et al., Angew. Chem. Inter. Edit. 1966, 5, 385; errata 511; Cahnet al., Angew. Chem. 1966, 78, 413; Cahn and Ingold, J. Chem. Soc. 1951(London), 612; Cahn et al., Experientia 1956, 12, 81; Cahn, J. Chem.Educ. 1964, 41, 116).

“Geometric isomer” means the diastereomers that owe their existence tohindered rotation about double bonds. These configurations aredifferentiated in their names by the prefixes cis and trans, or Z and E,which indicate that the groups are on the same or opposite side of thedouble bond in the molecule according to the Cahn-Ingold-Prelog rules.

Furthermore, the structures and other compounds discussed in thisapplication include all atropic isomers thereof. “Atropic isomers” are atype of stereoisomer in which the atoms of two isomers are arrangeddifferently in space. Atropic isomers owe their existence to arestricted rotation caused by hindrance of rotation of large groupsabout a central bond. Such atropic isomers typically exist as a mixture,however as a result of recent advances in chromatography techniques; ithas been possible to separate mixtures of two atropic isomers in selectcases.

“Tautomer” is one of two or more structural isomers that exist inequilibrium and is readily converted from one isomeric form to another.This conversion results in the formal migration of a hydrogen atomaccompanied by a switch of adjacent conjugated double bonds. Tautomersexist as a mixture of a tautomeric set in solution. In solid form,usually one tautomer predominates. In solutions where tautomerization ispossible, a chemical equilibrium of the tautomers will be reached. Theexact ratio of the tautomers depends on several factors, includingtemperature, solvent and pH. The concept of tautomers that areinterconvertable by tautomerizations is called tautomerism.

Of the various types of tautomerism that are possible, two are commonlyobserved. In keto-enol tautomerism a simultaneous shift of electrons anda hydrogen atom occurs. Ring-chain tautomerism arises as a result of thealdehyde group (—CHO) in a sugar chain molecule reacting with one of thehydroxy groups (—OH) in the same molecule to give it a cyclic(ring-shaped) form as exhibited by glucose. Common tautomeric pairs are:ketone-enol, amide-nitrile, lactam-lactim, amide-imidic acid tautomerismin heterocyclic rings (e.g., in nucleobases such as guanine, thymine andcytosine), amine-enamine and enamine-enamine. The compounds of thisapplication may also be represented in multiple tautomeric forms, insuch instances, the application expressly includes all tautomeric formsof the compounds described herein (e.g., alkylation of a ring system mayresult in alkylation at multiple sites, the application expresslyincludes all such reaction products).

In the present application, the structural formula of the bifunctionalcompound represents a certain isomer for convenience in some cases, butthe present application includes all isomers, such as geometricalisomers, optical isomers based on an asymmetrical carbon, stereoisomers,tautomers, and the like. In the present specification, the structuralformula of the compound represents a certain isomer for convenience insome cases, but the present application includes all isomers, such asgeometrical isomers, optical isomers based on an asymmetrical carbon,stereoisomers, tautomers, and the like.

Additionally, the compounds of the present application, for example, thesalts of the bifunctional compounds, can exist in either hydrated orunhydrated (the anhydrous) form or as solvates with other solventmolecules. Non-limiting examples of hydrates include monohydrates,dihydrates, etc. Non-limiting examples of solvates include ethanolsolvates, acetone solvates, etc.

“Solvate” means solvent addition forms that contain eitherstoichiometric or non stoichiometric amounts of solvent. Some compoundshave a tendency to trap a fixed molar ratio of solvent molecules in thecrystalline solid state, thus forming a solvate. If the solvent is waterthe solvate formed is a hydrate; and if the solvent is alcohol, thesolvate formed is an alcoholate. Hydrates are formed by the combinationof one or more molecules of water with one molecule of the substance inwhich the water retains its molecular state as H₂O.

The synthesized bifunctional compounds can be separated from a reactionmixture and further purified by a method such as column chromatography,high pressure liquid chromatography, or recrystallization. As can beappreciated by the skilled artisan, further methods of synthesizing thebifunctional compounds of the formulae herein will be evident to thoseof ordinary skill in the art. Additionally, the various synthetic stepsmay be performed in an alternate sequence or order to give the desiredcompounds. In addition, the solvents, temperatures, reaction durations,etc. delineated herein are for purposes of illustration only and one ofordinary skill in the art will recognize that variation of the reactionconditions can produce the desired bridged macrocyclic products of thepresent application. Synthetic chemistry transformations and protectinggroup methodologies (protection and deprotection) useful in synthesizingthe compounds described herein are known in the art and include, forexample, those such as described in R. Larock, Comprehensive OrganicTransformations, VCH Publishers (1989); T. W. Greene and P. G. M. Wuts,Protective Groups in Organic Synthesis, 2d. Ed., John Wiley and Sons(1991); L. Fieser and M. Fieser, Fieser and Fieser's Reagents forOrganic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed.,Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons(1995), and subsequent editions thereof.

The compounds of this application may be modified by appending variousfunctionalities via any synthetic means delineated herein to enhanceselective biological properties. Such modifications are known in the artand include those which increase biological penetration into a givenbiological system (e.g., blood, lymphatic system, central nervoussystem), increase oral availability, increase solubility to allowadministration by injection, alter metabolism and alter rate ofexcretion.

The compounds of the application are defined herein by their chemicalstructures and/or chemical names. Where a compound is referred to byboth a chemical structure and a chemical name, and the chemicalstructure and chemical name conflict, the chemical structure isdeterminative of the compound's identity.

The recitation of a listing of chemical groups in any definition of avariable herein includes definitions of that variable as any singlegroup or combination of listed groups. The recitation of an embodimentfor a variable herein includes that embodiment as any single embodimentor in combination with any other embodiments or portions thereof.

Method of Synthesizing the Compounds

Compounds of the present application can be prepared in a variety ofways using commercially available starting materials, compounds known inthe literature, or from readily prepared intermediates, by employingstandard synthetic methods and procedures either known to those skilledin the art, or which will be apparent to the skilled artisan in light ofthe teachings herein. Standard synthetic methods and procedures for thepreparation of organic molecules and functional group transformationsand manipulations can be obtained from the relevant scientificliterature or from standard textbooks in the field. Although not limitedto any one or several sources, classic texts such as Smith, M. B.,March, J., March's Advanced Organic Chemistry: Reactions, Mechanisms,and Structure, 5^(th) edition, John Wiley & Sons: New York, 2001; andGreene, T. W., Wuts, P. G. M., Protective Groups in Organic Synthesis,3^(rd) edition, John Wiley & Sons: New York, 1999, incorporated byreference herein, are useful and recognized reference textbooks oforganic synthesis known to those in the art. The following descriptionsof synthetic methods are designed to illustrate, but not to limit,general procedures for the preparation of compounds of the presentapplication. The processes generally provide the desired final compoundat or near the end of the overall process, although it may be desirablein certain instances to further convert the compound to apharmaceutically acceptable salt, ester or prodrug thereof. Suitablesynthetic routes are depicted in the schemes below.

Those skilled in the art will recognize if a stereocenter exists in thecompounds disclosed herein. Accordingly, the present applicationincludes both possible stereoisomers (unless specified in the synthesis)and includes not only racemic compounds but the individual enantiomersand/or diastereomers as well. When a compound is desired as a singleenantiomer or diastereomer, it may be obtained by stereospecificsynthesis or by resolution of the final product or any convenientintermediate. Resolution of the final product, an intermediate, or astarting material may be affected by any suitable method known in theart. See, for example, “Stereochemistry of Organic Compounds” by E. L.Eliel, S. H. Wilen, and L. N. Mander (Wiley-Interscience, 1994).

The compounds of the present application can be prepared in a number ofways well known to those skilled in the art of organic synthesis. By wayof example, compounds of the present application can be synthesizedusing the methods described below, together with synthetic methods knownin the art of synthetic organic chemistry, or variations thereon asappreciated by those skilled in the art. Preferred methods include butare not limited to those methods described below.

Compounds of the present application can be synthesized by following thesteps outlined in General Schemes 1-4 which comprise different sequencesof assembling intermediates 1a, 1b, 1c, 1d, 1e, 1f, 1g, 1h, 1i, 1j, 1k,1m, 1n, 1p, 1q, and 1r. Starting materials are either commerciallyavailable or made by known procedures in the reported literature or asillustrated.

wherein R¹, R², R³, A, X, and n are defined herein.

Target Ligand Compound of Formula TL-I can be synthesized usingintermediates 1a, 1b, 1c, 1d, 1e, 1f, and 1g is outlined in GeneralScheme 1. Cyclization of 1a and 1b using an acid, e.g., acetic acid, insolvent, e.g., CHCl₃, at elevated temperatures provides intermediate 1d.Alternatively, Intermediate 1d can be obtained via cyclization of 1a and1c using a base, e.g., N,N-diisopropylethylamine (DIEA) in a solvent,e.g., dimethylformamide (DMF) at elevated temperatures. Hydrolysis ofIntermediate 1d using a base, e.g., lithium hydroxide (LiOH) in asolvent, e.g., tetrahydrofuran (THF), methanol (MeOH), and/or water(1120), provides Ie. Coupling of acid Ie and amine If under standardcoupling conditions using a coupling reagent, e.g.,[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxidehexafluoro-phosphate (HATU), orO-benzotriazole-N,N,N′,N′-tetramethyl-uronium-hexafluoro-phosphate(HBTU), and a base, e.g., triethylamine or N,N-diisopropylethylamine(DIEA), in a solvent, e.g., dichloromethane or DMF, provides 1g. Suzukicoupling of 1g and 1h in the presence of a metal catalyst, i.e.,[1,1′-Bis(diphenylphosphino)ferrocene]dichloropalladium(II)(PdCl₂(dppf)₂), a phosphine ligand, i.e.,2-Dicyclohexylphosphino-2′,4′,6′-triisopropylbiphenyl (Xphos), and abase, i.e., sodium carbonate (Na₂CO₃) or potassium carbonate (K₂CO₃), ina solvent, i.e., dioxane, provides 1i. Deprotection of 1i using a strongacid, i.e., trifluoroacetic acid (TFA) or hydrochloric acid (HCl), in asolvent, i.e., dichloromethane (DCM) or dioxane, provides the desiredcompound of Formula TL-I.

wherein R¹, R², R³, A, X, and n are defined herein.

Target Ligand Compound of Formula TL-I can be synthesized usingintermediates 1i, 1p, and 1q is outlined in General Scheme 1. Alkylationof 1i with 1p in the presence of a base, i.e., K₂CO₃, and in a solvent,i.e., dimethylformamide (DMF), provides intermediate 1q. Deprotection of1q using a strong acid, i.e., trifluoroacetic acid (TFA) or hydrochloricacid (HCl), in a solvent, i.e., dichloromethane (DCM) or dioxane,provides the desired compound of Formula TL-I.

wherein R¹, R², R³, R¹³, R¹⁴, R¹⁵, R¹⁶, A, X, W, Z₁, p1, p2, p3, n, q,and v are defined herein.

The general way of preparing representative compounds of the presentapplication (i.e., Compound of Formula (I) shown above) usingintermediates 1i, 1j, 1k, 1m, and 1n is outlined in General Scheme 3.Amination of 1j with 1k in the presence of a base, i.e.,N,N-diisopropylethylamine (DIEA), in a solvent, i.e., DMF, at elevatedtemperatures provides intermediate 1m. Deprotection of 1m using a strongacid, i.e., trifluoroacetic acid (TFA) or hydrochloric acid (HCl), in asolvent, i.e., dichloromethane (DCM) or dioxane, provides carboxylicacid 1n. Coupling of acid in and amine TL-I under standard couplingconditions using a coupling reagent, e.g.,1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDCI), and a base, e.g.,DIEA, in a solvent, e.g., DCM or DMF, provides compound Formula (I).

wherein R¹, R², R³, R¹³, R¹⁴, R¹⁵, R¹⁶, A, X, W, Z, Z₁, p1, p2, p3, n,q, and v are defined herein.

Alternatively, compounds of the present application (i.e., Compound ofFormula (I) shown above) can be prepared using intermediates 1r and 1aas outlined in General Scheme 4. Coupling of acid 1q and amine 1r understandard coupling conditions using a coupling reagent, e.g.,[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxidehexafluoro-phosphate (HATU), orO-benzotriazole-N,N,N′,N′-tetramethyl-uronium-hexafluoro-phosphate(HBTU), and a base, e.g., triethylamine or DIEA, in a solvent, e.g.,dichloromethane or DMF, provides desired compound of Formula (I).

Biological Assays Biochemical Assays

EGFR biochemical assays are carried out using a homogeneoustime-resolved fluorescence (HTRF) assay. The reaction mixtures containbiotin-Lck-peptide substrate, wild type, or mutant EGFR enzyme inreaction buffer. Enzyme concentrations are adjusted to accommodatevarying kinase activity and ATP concentrations. Compounds of the presentapplication are diluted into the assay mixture and IC₅₀ values aredetermined using 12-point inhibition curves.

Phospho-EGFR Target Modulation Assays and ELISA

Cells are lysed with lysis buffer containing protease and phosphataseinhibitors and the plates are shaken. An aliquot from each well is thentransferred to prepared ELISA plates for analysis. Once harvested andplated, the cells are pre-treated with media with or without EGF. Thecompounds of the present application are then added and IC₅₀ values aredetermined using an EGFR biochemical assay described above.

Solid high-binding ELISA plates are coated with goat anti-EGFR captureantibody. Plates are then blocked with BSA in a buffer, and then washed.Aliquots of lysed cell are added to each well of the ELISA plate and theplate is incubated. An anti-phospho-EGFR is then added and is followedby further incubation. After washing, anti-rabbit-HRP is added and theplate is again incubated. Chemiluminescent detection is carried out withSuperSignal ELISA Pico substrate. Signal is read on EnVision platereader using built-in UltraLUM setting.

Western Blotting

Cell lysates are equalized to protein content and loaded onto a gel withrunning buffer. Membranes are probed with primary antibodies and arethen washed. HRP-conjugated secondary antibodies are added and afterwashing. HRP is detected using a HRP substrate reagent and recorded withan imager.

EGFR protein degradation is assessed by western blotting after treatmentof cell lines with a compound of the present applicationdose-dependently or combination with cetuximab.

Cell Proliferation Assays

Cell lines are plated in media. The compounds of the present applicationare then serially diluted and transferred to the cells. Cell viabilityis measured via a luminescent readout. Data is analyzed by non-linearregression curve-fitting.

Methods of the Application

In another aspect, the application provides a method of modulating(e.g., decreasing) the amount of a kinase, comprising contacting thekinase with a bifunctional compound disclosed herein, or apharmaceutically acceptable salt, hydrate, solvate, prodrug,stereoisomer, or tautomer thereof. In some embodiments, the kinasecomprises a mutated cysteine residue. In further embodiments, themutated cysteine residue is located in or near the position equivalentto Cys 797 in EGFR, including such position in Jak3, Blk, Bmx, Btk, HER2(ErbB2), HER4 (ErbB4), Itk, Tec, and Txk.

In another aspect, the application provides a method of modulating(e.g., decreasing) the amount of a kinase, comprising administering to asubject in need thereof an effective amount of a bifunctional compounddisclosed herein, or a pharmaceutically acceptable salt, hydrate,solvate, prodrug, stereoisomer, or tautomer thereof. In someembodiments, the kinase is EGFR or a mutant thereof. In otherembodiments, the kinase is a Her-kinase.

In still another aspect, the application provides a method of inhibitingepidermal growth factor receptor (EGFR), the method comprisingadministering to a subject in need thereof an effective amount of abifunctional compound disclosed herein, or a pharmaceutically acceptablesalt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.

Another aspect of the application provides a method of treating orpreventing a disease, the method comprising administering to a subjectin need thereof an effective amount of a bifunctional compound disclosedherein, or a pharmaceutically acceptable salt, hydrate, solvate,prodrug, stereoisomer, or tautomer thereof. In some embodiments, thedisease is mediated by a kinase. In further embodiments, the kinasecomprises a mutated cysteine residue. In further embodiments, themutated cysteine residue is located in or near the position equivalentto Cys 797 in EGFR, including such positions in Jak3, Blk, Bmx, Btk,HER2 (ErbB2), HER4 (ErbB4), Itk, Tec, and Txk.

In some embodiments, the disease is mediated by EGFR (e.g., EGFR plays arole in the initiation or development of the disease). In furtherembodiments, the EGFR is a Her-kinase. In further embodiments, theHer-kinase is HER1, HER2, or HER4.

In certain embodiments, the disease or disorder is cancer or aproliferation disease.

In further embodiments, the disease or disorder is lung cancer, coloncancer, breast cancer, prostate cancer, liver cancer, pancreas cancer,brain cancer, kidney cancer, ovarian cancer, stomach cancer, skincancer, bone cancer, gastric cancer, breast cancer, pancreatic cancer,glioma, glioblastoma, hepatocellular carcinoma, papillary renalcarcinoma, head and neck squamous cell carcinoma, leukemias, lymphomas,myelomas, or solid tumors.

In other embodiments, the disease or disorder is inflammation,arthritis, rheumatoid arthritis, spondyiarthropathies, gouty arthritis,osteoarthritis, juvenile arthritis, and other arthritic conditions,systemic lupus erthematosus (SLE), skin-related conditions, psoriasis,eczema, burns, dermatitis, neuroinflammation, allergy, pain, neuropathicpain, fever, pulmonary disorders, lung inflammation, adult respiratorydistress syndrome, pulmonary sarcoisosis, asthma, silicosis, chronicpulmonary inflammatory disease, and chronic obstructive pulmonarydisease (COPD), cardiovascular disease, arteriosclerosis, myocardialinfarction (including post-myocardial infarction indications),thrombosis, congestive heart failure, cardiac reperfusion injury, aswell as complications associated with hypertension and/or heart failuresuch as vascular organ damage, restenosis, cardiomyopathy, strokeincluding ischemic and hemorrhagic stroke, reperfusion injury, renalreperfusion injury, ischemia including stroke and brain ischemia, andischemia resulting from cardiac/coronary bypass, neurodegenerativedisorders, liver disease and nephritis, gastrointestinal conditions,inflammatory bowel disease, Crohn's disease, gastritis, irritable bowelsyndrome, ulcerative colitis, ulcerative diseases, gastric ulcers, viraland bacterial infections, sepsis, septic shock, gram negative sepsis,malaria, meningitis, HIV infection, opportunistic infections, cachexiasecondary to infection or malignancy, cachexia secondary to acquiredimmune deficiency syndrome (AIDS), AIDS, ARC (AIDS related complex),pneumonia, herpes virus, myalgias due to infection, influenza,autoimmune disease, graft vs. host reaction and allograft rejections,treatment of bone resorption diseases, osteoporosis, multiple sclerosis,cancer, leukemia, lymphoma, colorectal cancer, brain cancer, bonecancer, epithelial call-derived neoplasia (epithelial carcinoma), basalcell carcinoma, adenocarcinoma, gastrointestinal cancer, lip cancer,mouth cancer, esophageal cancer, small bowel cancer, stomach cancer,colon cancer, liver cancer, bladder cancer, pancreas cancer, ovariancancer, cervical cancer, lung cancer, breast cancer, skin cancer,squamous cell and/or basal cell cancers, prostate cancer, renal cellcarcinoma, and other known cancers that affect epithelial cellsthroughout the body, chronic myelogenous leukemia (CML), acute myeloidleukemia (AML) and acute promyelocytic leukemia (APL), angiogenesisincluding neoplasia, metastasis, central nervous system disorders,central nervous system disorders having an inflammatory or apoptoticcomponent, Alzheimer's disease, Parkinson's disease, Huntington'sdisease, amyotrophic lateral sclerosis, spinal cord injury, andperipheral neuropathy, or B-Cell Lymphoma.

In further embodiments, the disease or disorder is inflammation,arthritis, rheumatoid arthritis, spondylarthropathies, gouty arthritis,osteoarthritis, juvenile arthritis, and other arthritic conditions,systemic lupus erthematosus (SLE), skin-related conditions, psoriasis,eczema, dermatitis, pain, pulmonary disorders, lung inflammation, adultrespiratory distress syndrome, pulmonary sarcoisosis, asthma, chronicpulmonary inflammatory disease, and chronic obstructive pulmonarydisease (COPD), cardiovascular disease, arteriosclerosis, myocardialinfarction (including post-myocardial infarction indications),congestive heart failure, cardiac reperfusion injury, inflammatory boweldisease, Crohn's disease, gastritis, irritable bowel syndrome, leukemiaor lymphoma.

Another aspect of the application provides a method of treating a kinasemediated disorder, the method comprising administering to a subject inneed thereof an effective amount of a bifunctional compound disclosedherein, or a pharmaceutically acceptable salt, hydrate, solvate,prodrug, stereoisomer, or tautomer thereof. In some embodiments, thebifunctional is capable of modulating (e.g., decreasing) the amount ofEGFR. In other embodiments, the bifunctional compound is capable ofmodulating (e.g., decreasing) the amount of HER1, HER2, or HER4. Inother embodiments, the subject is administered an additional therapeuticagent. In other embodiments, the bifunctional compound and theadditional therapeutic agent are administered simultaneously orsequentially.

In other embodiments, the disease or disorder is cancer. In furtherembodiments, the cancer is lung cancer, colon cancer, breast cancer,prostate cancer, liver cancer, pancreas cancer, brain cancer, kidneycancer, ovarian cancer, stomach cancer, skin cancer, bone cancer,gastric cancer, breast cancer, pancreatic cancer, glioma, glioblastoma,hepatocellular carcinoma, papillary renal carcinoma, head and necksquamous cell carcinoma, leukemias, lymphomas, myelomas, or solidtumors.

Another aspect of the present application includes a method ofmodulating (e.g., decreasing) the amount of epidermal growth factorreceptor (EGFR). The method comprises administering to a subject in needthereof an effective amount of a bifunctional compound of theapplication or a pharmaceutically acceptable salt, hydrate, solvate,prodrug, stereoisomer, or tautomer thereof, or a pharmaceuticalcomposition of the application. In some embodiments, the bifunctionalcompound prevents EGFR dimer formation.

Another aspect of the present application describes a method of treatingor preventing a disease, wherein the disease is resistant to an EGFRtargeted therapy, such as a therapy with gefitinib, erlotinib, AZD9291,CO-1686 or WZ4002. The method comprises administering to a subject inneed thereof an effective amount of a bifunctional compound of theapplication, or a pharmaceutically acceptable salt, hydrate, solvate,prodrug, stereoisomer, or tautomer thereof.

In another aspect, the application provides a method of treating orpreventing cancer, wherein the cancer cell comprises activated EGFR,comprising administering to a subject in need thereof an effectiveamount of a bifunctional compound disclosed herein, or apharmaceutically acceptable salt, hydrate, solvate, prodrug,stereoisomer, or tautomer thereof.

In certain embodiments, the EGFR activation is selected from mutation ofEGFR, amplification of EGFR, expression of EGFR, and ligand mediatedactivation of EGFR.

In further embodiments, the mutation of EGFR is located at G719S, G719C,G719A, L858R, L861Q, an exon 19 deletion mutation, or an exon 20insertion mutation.

Another aspect of the application provides a method of treating orpreventing cancer in a subject, wherein the subject is identified asbeing in need of EGFR inhibition for the treatment of cancer, comprisingadministering to the subject an effective amount of a bifunctionalcompound disclosed herein, or a pharmaceutically acceptable salt,hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.

In certain embodiments, the subject identified as being in need of EGFRinhibition is resistant to a known EGFR inhibitor, including but notlimited to, gefitinib or erlotinib. In certain embodiments, a diagnostictest is performed to determine if the subject has an activating mutationin EGFR. In certain embodiments, a diagnostic test is performed todetermine if the subject has an EGFR harboring an activating and a drugresistance mutation. Activating mutations comprise without limitationL858R, G719S, G719C, G719A, L718Q, L861Q, a deletion in exon 19 and/oran insertion in exon 20. Drug resistant EGFR mutants can have withoutlimitation a drug resistance mutation comprising T790M, T854A, L718Q,C797S, or D761Y. The diagnostic test can comprise sequencing,pyrosequencing, PCR, RT-PCR, or similar analysis techniques known tothose of skill in the art that can detect nucleotide sequences.

In another aspect, the application provides a method of treating orpreventing cancer, wherein the cancer cell comprises an activated ERBB2,comprising administering to a subject in need thereof an effectiveamount of a bifunctional compound disclosed herein, or apharmaceutically acceptable salt, hydrate, solvate, prodrug,stereoisomer, or tautomer thereof. In certain embodiments, the ERBB2activation is selected from mutation of ERBB2, expression of ERBB2, andamplification of ERBB2. In further embodiments, the mutation is amutation in exon 20 of ERBB2.

In another aspect, the application provides a method of treating cancerin a subject, wherein the subject is identified as being in need ofERBB2 inhibition for the treatment of cancer, comprising administeringto the subject in need thereof an effective amount of a bifunctionalcompound disclosed herein, or a pharmaceutically acceptable salt,hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.

Another aspect of the application provides a method of preventingresistance to a known EGFR inhibitor, including but not limited to,gefitinib or erlotinib in a disease, comprising administering to asubject in need thereof an effective amount of a bifunctional compounddisclosed herein, or a pharmaceutically acceptable salt, hydrate,solvate, prodrug, stereoisomer, or tautomer thereof.

In certain embodiments, the application provides a method of treatingany of the disorders described herein, wherein the subject is a human.In certain embodiments, the application provides a method of preventingany of the disorders described herein, wherein the subject is a human.

In another aspect, the application provides a bifunctional compounddisclosed herein, or a pharmaceutically acceptable salt, hydrate,solvate, prodrug, stereoisomer, or tautomer thereof, for use in themanufacture of a medicament for treating or preventing a disease inwhich EGFR plays a role.

In still another aspect, the application provides the a bifunctionalcompound of the application, or a pharmaceutically acceptable salt,hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, or apharmaceutical composition of the application for use in the treatmentor prevention of a disease in which EGFR plays a role.

The bifunctional compounds and compositions of this application areparticularly useful for treating or lessening the severity of a disease,condition, or disorder where a protein kinase is implicated in thedisease, condition, or disorder. In one aspect, the present applicationprovides a method for treating or lessening the severity of a disease,condition, or disorder where a protein kinase is implicated in thedisease state. In another aspect, the present application provides amethod for treating or lessening the severity of a kinase disease,condition, or disorder where inhibition of enzymatic activity isimplicated in the treatment of the disease. In another aspect, thisapplication provides a method for treating or lessening the severity ofa disease, condition, or disorder with bifunctional compounds thatinhibit enzymatic activity by binding to the protein kinase. Anotheraspect provides a method for treating or lessening the severity of akinase disease, condition, or disorder by inhibiting enzymatic activityof the kinase with a protein kinase inhibitor.

In some embodiments, said method is used to treat or prevent a conditionselected from autoimmune diseases, inflammatory diseases, proliferativeand hyperproliferative diseases, immunologically-mediated diseases, bonediseases, metabolic diseases, neurological and neurodegenerativediseases, cardiovascular diseases, hormone related diseases, allergies,asthma, and Alzheimer's disease. In other embodiments, said condition isselected from a proliferative disorder and a neurodegenerative disorder.

One aspect of this application provides bifunctional compounds that areuseful for the treatment of diseases, disorders, and conditionscharacterized by excessive or abnormal cell proliferation. Such diseasesinclude, but are not limited to, a proliferative or hyperproliferativedisease, and a neurodegenerative disease. Examples of proliferative andhyperproliferative diseases include, without limitation, cancer. Theterm “cancer” includes, but is not limited to, the following cancers:breast; ovary; cervix; prostate; testis, genitourinary tract; esophagus;larynx, glioblastoma; neuroblastoma; stomach; skin, keratoacanthoma;lung, epidermoid carcinoma, large cell carcinoma, small cell carcinoma,lung adenocarcinoma; bone; colon; colorectal; adenoma; pancreas,adenocarcinoma; thyroid, follicular carcinoma, undifferentiatedcarcinoma, papillary carcinoma; seminoma; melanoma; sarcoma; bladdercarcinoma; liver carcinoma and biliary passages; kidney carcinoma;myeloid disorders; lymphoid disorders, Hodgkin's, hairy cells; buccalcavity and pharynx (oral), lip, tongue, mouth, pharynx; small intestine;colonrectum, large intestine, rectum, brain and central nervous system;chronic myeloid leukemia (CML), and leukemia. The term “cancer”includes, but is not limited to, the following cancers: myeloma,lymphoma, or a cancer selected from gastric, renal, or and the followingcancers: head and neck, oropharangeal, non-small cell lung cancer(NSCLC), endometrial, hepatocarcinoma, Non-Hodgkins lymphoma, andpulmonary.

The term “cancer” refers to any cancer caused by the proliferation ofmalignant neoplastic cells, such as tumors, neoplasms, carcinomas,sarcomas, leukemias, lymphomas and the like. For example, cancersinclude, but are not limited to, mesothelioma, leukemias and lymphomassuch as cutaneous T-cell lymphomas (CTCL), noncutaneous peripheralT-cell lymphomas, lymphomas associated with human T-cell lymphotrophicvirus (HTLV) such as adult T-cell leukemia/lymphoma (ATLL), B-celllymphoma, acute nonlymphocytic leukemias, chronic lymphocytic leukemia,chronic myelogenous leukemia, acute myelogenous leukemia, lymphomas, andmultiple myeloma, non-Hodgkin lymphoma, acute lymphatic leukemia (ALL),chronic lymphatic leukemia (CLL), Hodgkin's lymphoma, Burkitt lymphoma,adult T-cell leukemia lymphoma, acute-myeloid leukemia (AML), chronicmyeloid leukemia (CML), or hepatocellular carcinoma. Further examplesinclude myelodisplastic syndrome, childhood solid tumors such as braintumors, neuroblastoma, retinoblastoma, Wilms' tumor, bone tumors, andsoft-tissue sarcomas, common solid tumors of adults such as head andneck cancers (e.g., oral, laryngeal, nasopharyngeal and esophageal),genitourinary cancers (e.g., prostate, bladder, renal, uterine, ovarian,testicular), lung cancer (e.g., small-cell and non-small cell), breastcancer, pancreatic cancer, melanoma and other skin cancers, stomachcancer, brain tumors, tumors related to Gorlin's syndrome (e.g.,medulloblastoma, meningioma, etc.), and liver cancer. Additionalexemplary forms of cancer which may be treated by the subjectbifunctional compounds include, but are not limited to, cancer ofskeletal or smooth muscle, stomach cancer, cancer of the smallintestine, rectum carcinoma, cancer of the salivary gland, endometrialcancer, adrenal cancer, anal cancer, rectal cancer, parathyroid cancer,and pituitary cancer.

Additional cancers that the bifunctional compounds described herein maybe useful in preventing, treating and studying are, for example, coloncarcinoma, familiary adenomatous polyposis carcinoma and hereditarynon-polyposis colorectal cancer, or melanoma. Further, cancers include,but are not limited to, labial carcinoma, larynx carcinoma, hypopharynxcarcinoma, tongue carcinoma, salivary gland carcinoma, gastriccarcinoma, adenocarcinoma, thyroid cancer (medullary and papillarythyroid carcinoma), renal carcinoma, kidney parenchyma carcinoma, cervixcarcinoma, uterine corpus carcinoma, endometrium carcinoma, chorioncarcinoma, testis carcinoma, urinary carcinoma, melanoma, brain tumorssuch as glioblastoma, astrocytoma, meningioma, medulloblastoma andperipheral neuroectodermal tumors, gall bladder carcinoma, bronchialcarcinoma, multiple myeloma, basalioma, teratoma, retinoblastoma,choroidea melanoma, seminoma, rhabdomyosarcoma, craniopharyngeoma,osteosarcoma, chondrosarcoma, myosarcoma, liposarcoma, fibrosarcoma,Ewing sarcoma, and plasmocytoma. In one aspect of the application, thepresent application provides for the use of one or more bifunctionalcompounds of the application in the manufacture of a medicament for thetreatment of cancer, including without limitation the various types ofcancer disclosed herein.

In some embodiments, the bifunctional compounds of this application areuseful for treating cancer, such as colorectal, thyroid, breast, andlung cancer; and myeloproliferative disorders, such as polycythemiavera, thrombocythemia, myeloid metaplasia with myelofibrosis, chronicmyelogenous leukemia, chronic myelomonocytic leukemia, hypereosinophilicsyndrome, juvenile myelomonocytic leukemia, and systemic mast celldisease. In some embodiments, the bifunctional compounds of thisapplication are useful for treating hematopoietic disorders, inparticular, acute-myelogenous leukemia (AML), chronic-myelogenousleukemia (CML), acute-promyelocytic leukemia, and acute lymphocyticleukemia (ALL).

This application further embraces the treatment or prevention of cellproliferative disorders such as hyperplasias, dysplasias andpre-cancerous lesions. Dysplasia is the earliest form of pre-cancerouslesion recognizable in a biopsy by a pathologist. The subjectbifunctional compounds may be administered for the purpose of preventingsaid hyperplasias, dysplasias or pre-cancerous lesions from continuingto expand or from becoming cancerous. Examples of pre-cancerous lesionsmay occur in skin, esophageal tissue, breast and cervicalintra-epithelial tissue.

Examples of neurodegenerative diseases include, without limitation,Adrenoleukodystrophy (ALD), Alexander's disease, Alper's disease,Alzheimer's disease, Amyotrophic lateral sclerosis (Lou Gehrig'sDisease), Ataxia telangiectasia, Batten disease (also known asSpielmeyer-Vogt-Sjogren-Batten disease), Bovine spongiformencephalopathy (BSE), Canavan disease, Cockayne syndrome, Corticobasaldegeneration, Creutzfeldt-Jakob disease, Familial fatal insomnia,Frontotemporal lobar degeneration, Huntington's disease, HIV-associateddementia, Kennedy's disease, Krabbe's disease, Lewy body dementia,Neuroborreliosis, Machado-Joseph disease (Spinocerebellar ataxia type3), Multiple System Atrophy, Multiple sclerosis, Narcolepsy, NiemannPick disease, Parkinson's disease, Pelizaeus-Merzbacher Disease, Pick'sdisease, Primary lateral sclerosis, Prion diseases, ProgressiveSupranuclear Palsy, Refsum's disease, Sandhoff disease, Schilder'sdisease, Subacute combined degeneration of spinal cord secondary toPernicious Anaemia, Spielmeyer-Vogt-Sjogren-Batten disease (also knownas Batten disease), Spinocerebellar ataxia (multiple types with varyingcharacteristics), Spinal muscular atrophy, Steele-Richardson-Olszewskidisease, Tabes dorsalis, and Toxic encephalopathy.

Another aspect of this application provides a method for the treatmentor lessening the severity of a disease selected from a proliferative orhyperproliterative disease, or a neurodegenerative disease, comprisingadministering an effective amount of a bifunctional compound, or apharmaceutically acceptable composition comprising a bifunctionalcompound, to a subject in need thereof. In other embodiments, the methodfurther comprises administering a second agent wherein said second agentprevents EGFR dimer formation. In some embodiments, the second agentthat prevents EGFR dimer formation is an antibody. In furtherembodiments, the second agent that prevents EGFR dimer formation iscetuximab, trastuzumab, or panitumumab. In further embodiments, thesecond agent that prevents EGFR dimer formation is cetuximab.

The bifunctional compounds and compositions of this application are alsouseful in biological samples. One aspect of the application isinhibiting protein kinase activity in a biological sample, which methodcomprises contacting said biological sample with a bifunctional compoundof the application or a composition comprising said bifunctionalcompound. The term “biological sample”, as used herein, means an invitro or an ex vivo sample, including, without limitation, cell culturesor extracts thereof; biopsied material obtained from a mammal orextracts thereof; and blood, saliva, urine, feces, semen, tears, orother body fluids or extracts thereof. Inhibition of protein kinaseactivity in a biological sample is useful for a variety of purposes thatare known to one of skill in the art. Examples of such purposes include,but are not limited to, blood transfusion, organ-transplantation, andbiological specimen storage.

Another aspect of this application includes the study of Her kinases inbiological and pathological phenomena; the study of intracellular signaltransduction pathways mediated by such protein kinases; and thecomparative evaluation of new protein kinase inhibitors. Examples ofsuch uses include, but are not limited to, biological assays such asenzyme assays and cell-based assays.

The activity of the bifunctional compounds and compositions of thepresent application as Her kinase modulators (e.g., capable ofmodulating or decreasing the amount of Her kinase) may be assayed invitro, in vivo, or in a cell line. In vitro assays include assays thatdetermine inhibition of either the kinase activity or ATPase activity ofthe activated kinase. Alternate in vitro assays quantitate the abilityof the inhibitor to bind to the protein kinase and may be measuredeither by radio labelling the inhibitor prior to binding, isolating theinhibitor/kinase complex and determining the amount of radio labelbound, or by running a competition experiment where new inhibitors areincubated with the kinase bound to known radioligands. Detailedconditions for assaying a compound utilized in this application as aninhibitor of various kinases are set forth in the Examples below.

In accordance with the foregoing, the present application furtherprovides a method for preventing or treating any of the diseases ordisorders described above in a subject in need of such treatment, whichmethod comprises administering to said subject a therapeuticallyeffective amount of a bifunctional compound of the application, or apharmaceutically acceptable salt, hydrate, solvate, prodrug,stereoisomer, or tautomer thereof, and optionally a second agent whereinsaid second agent prevents EGFR dimer formation. For any of the aboveuses, the required dosage will vary depending on the mode ofadministration, the particular condition to be treated and the effectdesired.

Pharmaceutical Compositions

In another aspect, the application provides a pharmaceutical compositioncomprising a therapeutically effective amount of a bifunctional compoundof the present application or an enantiomer, diastereomer, stereoisomer,or pharmaceutically acceptable salt thereof, and a pharmaceuticallyacceptable carrier.

Bifunctional compounds of the application can be administered aspharmaceutical compositions by any conventional route, in particularenterally, e.g., orally, e.g., in the form of tablets or capsules, orparenterally, e.g., in the form of injectable solutions or suspensions,or topically, e.g., in the form of lotions, gels, ointments or creams,or in a nasal or suppository form. Pharmaceutical compositionscomprising a compound of the present application in free form or in apharmaceutically acceptable salt form in association with at least onepharmaceutically acceptable carrier or diluent can be manufactured in aconventional manner by mixing, granulating or coating methods. Forexample, oral compositions can be tablets or gelatin capsules comprisingthe active ingredient together with a) diluents, e.g., lactose,dextrose, sucrose, mannitol, sorbitol, cellulose and/or glycine; b)lubricants, e.g., silica, talcum, stearic acid, its magnesium or calciumsalt and/or polyethyleneglycol; for tablets also c) binders, e.g.,magnesium aluminum silicate, starch paste, gelatin, tragacanth,methylcellulose, sodium carboxymethylcellulose and orpolyvinylpyrrolidone; if desired d) disintegrants, e.g., starches, agar,alginic acid or its sodium salt, or effervescent mixtures; and/or e)absorbents, colorants, flavors and sweeteners. Injectable compositionscan be aqueous isotonic solutions or suspensions, and suppositories canbe prepared from fatty emulsions or suspensions. The compositions may besterilized and/or contain adjuvants, such as preserving, stabilizing,wetting or emulsifying agents, solution promoters, salts for regulatingthe osmotic pressure and/or buffers. In addition, they may also containother therapeutically valuable substances. Suitable formulations fortransdermal applications include an effective amount of a compound ofthe present application with a carrier. A carrier can include absorbablepharmacologically acceptable solvents to assist passage through the skinof the host. For example, transdermal devices are in the form of abandage comprising a backing member, a reservoir containing the compoundoptionally with carriers, optionally a rate controlling barrier todeliver the compound to the skin of the host at a controlled andpredetermined rate over a prolonged period of time, and means to securethe device to the skin. Matrix transdermal formulations may also beused. Suitable formulations for topical application, e.g., to the skinand eyes, are preferably aqueous solutions, ointments, creams or gelswell-known in the art. Such may contain solubilizers, stabilizers,tonicity enhancing agents, buffers and preservatives.

The pharmaceutical compositions of the present application comprise atherapeutically effective amount of a compound of the presentapplication formulated together with one or more pharmaceuticallyacceptable carriers. As used herein, the term “pharmaceuticallyacceptable carrier” means a non-toxic, inert solid, semi-solid or liquidfiller, diluent, encapsulating material or formulation auxiliary of anytype. Some examples of materials which can serve as pharmaceuticallyacceptable carriers include, but are not limited to, ion exchangers,alumina, aluminum stearate, lecithin, serum proteins, such as humanserum albumin, buffer substances such as phosphates, glycine, sorbicacid, or potassium sorbate, partial glyceride mixtures of saturatedvegetable fatty acids, water, salts or electrolytes, such as protaminesulfate, disodium hydrogen phosphate, potassium hydrogen phosphate,sodium chloride, zinc salts, colloidal silica, magnesium trisilicate,polyvinyl pyrrolidone, polyacrylates, waxes, polyethylenepolyoxypropylene-block polymers, wool fat, sugars such as lactose, glucose andsucrose; starches such as corn starch and potato starch; cellulose andits derivatives such as sodium carboxymethyl cellulose, ethyl celluloseand cellulose acetate; powdered tragacanth; malt; gelatin; talc;excipients such as cocoa butter and suppository waxes, oils such aspeanut oil, cottonseed oil; safflower oil; sesame oil; olive oil; cornoil and soybean oil; glycols such a propylene glycol or polyethyleneglycol; esters such as ethyl oleate and ethyl laurate, agar; bufferingagents such as magnesium hydroxide and aluminum hydroxide; alginic acid;pyrogen-free water, isotonic saline; Ringer's solution; ethyl alcohol,and phosphate buffer solutions, as well as other non-toxic compatiblelubricants such as sodium lauryl sulfate and magnesium stearate, as wellas coloring agents, releasing agents, coating agents, sweetening,flavoring and perfuming agents, preservatives and antioxidants can alsobe present in the composition, according to the judgment of theformulator.

The pharmaceutical compositions of this application can be administeredto humans and other animals orally, rectally, parenterally,intracisternally, intravaginally, intraperitoneally, topically (as bypowders, ointments, or drops), buccally, or as an oral or nasal spray.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, microemulsions, solutions, suspensions, syrups andelixirs. In addition to the active compounds, the liquid dosage formsmay contain inert diluents commonly used in the art such as, forexample, water or other solvents, solubilizing agents and emulsifierssuch as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethylacetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyleneglycol, dimethylformamide, oils (in particular, cottonseed, groundnut,corn, germ, olive, castor, and sesame oils), glycerol,tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid estersof sorbitan, and mixtures thereof. Besides inert diluents, the oralcompositions can also include adjuvants such as wetting agents,emulsifying and suspending agents, sweetening, flavoring, and perfumingagents.

Injectable preparations, for example, sterile injectable aqueous, oroleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectablesolution, suspension or emulsion in a nontoxic parenterally acceptablediluent or solvent, for example, as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables.

In order to prolong the effect of a drug, it is often desirable to slowthe absorption of the drug from subcutaneous or intramuscular injection.This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material with poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionwhich, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

Compositions for rectal or vaginal administration are preferablysuppositories which can be prepared by mixing the compounds of thisapplication with suitable non-irritating excipients or carriers such ascocoa butter, polyethylene glycol or a suppository wax which are solidat ambient temperature but liquid at body temperature and therefore meltin the rectum or vaginal cavity and release the active compound.

Solid compositions of a similar type may also be employed as fillers insoft and hard filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like.

The active compounds can also be in micro-encapsulated form with one ormore excipients as noted above. The solid dosage forms of tablets,dragees, capsules, pills, and granules can be prepared with coatings andshells such as enteric coatings, release controlling coatings and othercoatings well known in the pharmaceutical formulating art. In such soliddosage forms the active compound may be admixed with at least one inertdiluent such as sucrose, lactose or starch. Such dosage forms may alsocomprise, as is normal practice, additional substances other than inertdiluents, e.g., tableting lubricants and other tableting aids such amagnesium stearate and microcrystalline cellulose. In the case ofcapsules, tablets and pills, the dosage forms may also comprisebuffering agents.

Dosage forms for topical or transdermal administration of a compound ofthis application include ointments, pastes, creams, lotions, gels,powders, solutions, sprays, inhalants or patches. The active componentis admixed under sterile conditions with a pharmaceutically acceptablecarrier and any needed preservatives or buffers as may be required.Ophthalmic formulation, ear drops, eye ointments, powders and solutionsare also contemplated as being within the scope of this application.

The ointments, pastes, creams and gels may contain, in addition to anactive compound of this application, excipients such as animal andvegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulosederivatives, polyethylene glycols, silicones, bentonites, silicic acid,talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to the compounds of thisapplication, excipients such as lactose, talc, silicic acid, aluminumhydroxide, calcium silicates and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants suchas chlorofluorohydrocarbons.

Transdermal patches have the added advantage of providing controlleddelivery of a compound to the body. Such dosage forms can be made bydissolving or dispensing the compound in the proper medium. Absorptionenhancers can also be used to increase the flux of the compound acrossthe skin. The rate can be controlled by either providing a ratecontrolling membrane or by dispersing the compound in a polymer matrixor gel.

Compounds and compositions of the application can be administered intherapeutically effective amounts in a combinational therapy with one ormore therapeutic agents (pharmaceutical combinations) or modalities,e.g., a second agent wherein the second agent modulates (e.g., inhibits)one or more other EGFR and/or other anti-proliferative, anti-cancer,immunomodulatory or anti-inflammatory substances. Where the compounds ofthe application are administered in conjunction with other therapies,dosages of the co-administered compounds will of course vary dependingon the type of co-drug employed, on the specific drug employed, on thecondition being treated and so forth. Compounds and compositions of theapplication can be administered in therapeutically effective amounts ina combinational therapy with one or more therapeutic agents(pharmaceutical combinations) or modalities, e.g., a second agentwherein said second agent prevents EGFR dimer formation, non-drugtherapies, etc. For example, synergistic effects can occur with agentsthat prevents EGFR dimer formation, other anti-proliferative,anti-cancer, immunomodulatory or anti-inflammatory substances. Where thecompounds of the application are administered in conjunction with othertherapies, dosages of the co-administered compounds will of course varydepending on the type of co-drug employed, on the specific drugemployed, on the condition being treated and so forth.

Combination therapy includes the administration of the subject compoundsin further combination with one or more other biologically activeingredients (such as, but not limited to, a second agent wherein saidsecond agent prevents EGFR dimer formation, a second and differentantineoplastic agent, a second EGFR inhibitor) and non-drug therapies(such as, but not limited to, surgery or radiation treatment). Forinstance, the compounds of the application can be used in combinationwith other pharmaceutically active compounds, preferably compounds thatare able to enhance the effect of the compounds of the application. Thecompounds of the application can be administered simultaneously (as asingle preparation or separate preparation) or sequentially to the otherdrug therapy or treatment modality. In general, a combination therapyenvisions administration of two or more drugs during a single cycle orcourse of therapy.

In one aspect of the application, the compounds may be administered incombination with one or more agents that prevent EGFR dimer formation.In some embodiments, the second agent that prevents EGFR dimer formationis an antibody. In further embodiments, the second agent that preventsEGFR dimer formation is cetuximab, trastuzumab, or panitumumab. Infurther embodiments, the second agent that prevents EGFR dimer formationis cetuximab.

In another aspect of the application, the compounds may be administeredin combination with one or more separate pharmaceutical agents, e.g., achemotherapeutic agent, an immunotherapeutic agent, or an adjunctivetherapeutic agent. In one embodiment, the chemotherapeutic agent reducesor inhibits the binding of ATP with EGFR (e.g., gefitinib, erlotinib,AZD9291, CO-1686 or WZ4002).

Examples Analytical Methods, Materials, and Instrumentation

All reactions were monitored by Waters Acquity UPLC/MS system (WatersPDA eλ Detector, QDa Detector, Sample manager—FL, Binary SolventManager) using Acquity UPLC® BEH C18 column (2.1×50 mm, 1.7 μm particlesize): solvent gradient=90% A at 0 min, 1% A at 1.8 min; solvent A=0.1%formic acid in Water; solvent B=0.1% formic acid in Acetonitrile; flowrate: 0.6 mL/min. Reaction products were purified by flash columnchromatography using CombiFlash® Rf with Teledyne Isco RediSep® Rf HighPerformance Gold or Silicycle SiliaSep™ High Performance columns (4 g,12 g, 24 g, 40 g, or 80 g), Waters HPLC system using SunFire™ Prep C18column (19×100 mm, 5 μm particle size): solvent gradient=80% A at 0 min,5% A at 25 min; solvent A=0.035% TFA in Water; solvent B=0.035% TFA inMeOH; flow rate: 25 mL/min (Method A), and Waters Acquity UPLC/MS system(Waters PDA ex Detector, QDa Detector, Sample manager—FL, Binary SolventManager) using Acquity UPLC® BEH C18 column (2.1×50 mm, 1.7 m particlesize): solvent gradient=80% A at 0 min, 5% A at 2 min; solvent A=0.1%formic acid in Water; solvent B=0.1% formic acid in Acetonitrile; flowrate: 0.6 mL/min (method B). The purity of all compounds was over 95%and was analyzed with Waters LC/MS system. ¹H NMR was obtained using a500 MHz Bruker Avance III. Chemical shifts are reported relative todimethyl sulfoxide (δ=2.50) for ¹H NMR. Data are reported as (br=broad,s=singlet, d=doublet, t=triplet, q=quartet, m=multiplet).

Abbreviations Used in the Following Examples and Elsewhere Herein are:

-   -   atm atmosphere    -   br broad    -   DCM dichloromethane    -   DIEA N,N-diisopropylethylamine    -   DMA N,N-dimethylacetamide    -   DMF N,N-dimethylformamide    -   DMSO dimethyl sulfoxide    -   EDCI 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide    -   ESI electrospray ionization    -   EtOAc ethyl acetate    -   HCl hydrochloric acid    -   h hour(s)    -   HATU        bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium        3-oxide hexafluoro-phosphate    -   HPLC high-performance liquid chromatography    -   LCMS liquid chromatography-mass spectrometry    -   m multiplet    -   MeOH methanol    -   MHz megahertz    -   min minutes    -   MS mass spectrometry    -   NMR nuclear magnetic resonance    -   Pd₂(dba)₃ tris(dibenzylideneacetone)dipalladium(0)    -   ppm parts per million    -   TBAF tetra-n-butylammonium fluoride    -   THE tetrahydrofuran    -   TLC thin layer chromatography    -   Xphos 2-dicyclohexylphosphino-2′,4′,6′-triisopropylbiphenyl

Example 1: Synthesis of2-(1-Oxo-5-phenylisoindolin-2-yl)-2-phenyl-N-(thiazol-2-yl)acetamide2-(1-oxo-6-(4-(piperazin-1-yl)phenyl)isoindolin-2-yl)-2-phenyl-N-(thiazol-2-yl)acetamide(2-6)

Step 1. Methyl 2-(6-bromo-1-oxoisoindolin-2-yl)-2-phenylacetate (2-2)

To a solution of methyl 2-amino-2-phenylacetate (2-1, 2.8 g, 13.9 mmol)and methyl 5-bromo-2-(bromomethyl)benzoate (3.9 g, 12.7 mmol) inN,N-dimethylformamide (120 mL) was added DIEA (6.6 mL, 38.0 mmol) andthe resulting mixture was heated to 80° C. After stirring overnight, thereaction mixture was cooled down to room temperature and diluted withwater (700 mL). The precipitate was filtered off and dried using astream of nitrogen gas to obtain methyl2-(6-bromo-1-oxoisoindolin-2-yl)-2-phenylacetate (2-2) (3.2 g, 70%) asan off-white solid.

Step 2. 2-(6-Bromo-1-oxoisoindolin-2-yl)-2-phenylacetic acid (2-3)

To a solution of methyl 2-(6-bromo-1-oxoisoindolin-2-yl)-2-phenylacetate(2-2, 3.2 g, 8.89 mmol) in THF/MeOH/water (150 mL, 1:1:1) was addedlithium hydroxide monohydrate (2.65 g, 63.3 mmol). After stirring for 1h, the solvent was removed under reduced pressure and the resultingresidue was diluted with ice water. The aqueous mixture was acidifiedwith concentrated HCl and the resulting suspension isolated viafiltration. The solid was dried using a stream of nitrogen gas to obtain2-(6-bromo-1-oxoisoindolin-2-yl)-2-phenylacetic acid (2-3) (2.8 g, 92%)as an off-white solid.

Step 3.2-(6-Bromo-1-oxoisoindolin-2-yl)-2-phenyl-N-(thiazol-2-yl)acetamide(2-4) To a solution of methyl2-(6-bromo-1-oxoisoindolin-2-yl)-2-phenylacetic acid (2-3, 2.0 g, 5.78mmol), thiazol-2-amine (1.1 g, 11.6 mmol), and HATU (4.4 g, 11.6 mmol)in N,N-dimethylformamide (30 mL) was added DIEA (4.0 mL, 23.1 mmol).After stirring for 6 hr, the reaction mixture was diluted with EtOAc andwashed with water five times. The organic layer was dried over sodiumsulfate, filtered, concentrated under reduced pressure and purified bycolumn chromatography on silica gel (DCM:EtOAc=9:1 to 4:6) to obtain2-(6-bromo-1-oxoisoindolin-2-yl)-2-phenyl-N-(thiazol-2-yl)acetamide(2-4, 1.8 g, 73%) as an off-white solid.

Step 4. tert-Butyl4-(4-(3-oxo-2-(2-oxo-1-phenyl-2-(thiazol-2-ylamino)ethyl)isoindolin-5-yl)phenyl)piperazine-1-carboxylate(2-5)

A mixture of2-(6-bromo-1-oxoisoindolin-2-yl)-2-phenyl-N-(thiazol-2-yl)acetamide (50mg, 0.117 mmol),(4-(4-(tert-butoxycarbonyl)piperazin-1-yl)phenyl)boronic acid (21 mg,0.175 mmol) and 2 N Sodium carbonate (0.18 mL, 0.351 mmol) in dioxane (1mL) was degassed and heated to 100° C. PdCl₂(dppf)₂ (5 mg, 0.007 mmol)and Xphos (4.5 mg, 0.011 mmol) were then added and the resultingreaction mixture was stirred for 2 hr. The reaction mixture was thencooled down to room temperature and diluted with dichloromethane. Theresulting mixture was washed with water and brine, dried over sodiumsulfate, filtered, and concentrated under reduced pressure. Theresulting residue was purified by preparative high performance liquidchromatography (HPLC) to provide tert-butyl4-(4-(3-oxo-2-(2-oxo-1-phenyl-2-(thiazol-2-ylamino)ethyl)isoindolin-5-yl)phenyl)piperazine-1-carboxylate(2-5, 25 mg, 50%) as a white solid.

Step 5.2-(1-Oxo-5-phenylisoindolin-2-yl)-2-phenyl-N-(thiazol-2-yl)acetamidetrifluoroacetic acid salt (2-6)

To a solution of tert-butyl4-(4-(3-oxo-2-(2-oxo-1-phenyl-2-(thiazol-2-ylamino)ethyl)isoindolin-5-yl)phenyl)piperazine-1-carboxylate (2-5, 25 mg, 0.058mmol) in DCM (1.0 mL) was added trifluoroacetic acid (0.2 mL). Theresulting solution was stirred for 1 hr and then concentrated underreduced pressure. The resulting residue was purified by preparative highperformance liquid chromatography (HPLC) to provide 2-6. ¹H NMR 600 MHz(DMSO-d₆) δ 12.69 (bs, NH), 7.89 (d, J=1.3 Hz, 1H), 7.86 (dd, J=8.0, 1.7Hz, 1H), 7.65 (d, J=8.9 Hz, 2H), 7.60 (d, J=8.0 Hz, 1H), 7.49-7.45 (m,3H), 7.44-7.40 (m, 1H), 7.39-7.36 (m, 2H), 7.27 (d, J=3.5 Hz, 1H), 7.09(d, J=8.9 Hz, 2H), 6.31 (s, 1H), 4.76 (d, J=17.5 Hz, 1H), 3.99 (d,J=17.6 Hz, 1H), 3.42-3.39 (m, 4H), 3.25 (s, 4H). MS m/z: 510.28 [M+1].

Example 2: Synthesis of2-(2-((tert-Butyldimethylsilyl)oxy)-5-fluorophenyl)-2-(1-oxo-6-(4-(piperazin-1-yl)phenyl)isoindolin-2-yl)-N-(thiazol-2-yl)acetamide(2-13)

Step 1.2-(6-Bromo-1-oxoisoindolin-2-yl)-2-(5-fluoro-2-hydroxyphenyl)acetic acid(2-8)

To a solution of 2-amino-2-(5-fluoro-2-hydroxyphenyl)acetic acid (2-7)(250 mg, 1.13 mmol) and methyl 5-bromo-2-(bromomethyl)benzoate (331 mg,1.07 mmol) in dioxane (5 mL) was added DIEA (0.49 mL, 2.83 mmol) and theresulting mixture was stirred at 0° C. for 2 hr and then at roomtemperature for 2 hr. The reaction mixture was then warmed to 30° C. andstirred for an additional 4 hr at 30° C. The mixture was cooled down to0° C. and diluted with EtOAc. The resulting solution was washed with 1 NHCl solution and brine and the organic layer was dried over Na₂SO₄,filtered, and concentrated under reduced pressure to obtain2-(6-bromo-1-oxoisoindolin-2-yl)-2-(5-fluoro-2-hydroxyphenyl)acetic acid(2-8) which was used in the next step without further purification.

Step 2.2-(6-Bromo-1-oxoisoindolin-2-yl)-2-(5-fluoro-2-hydroxyphenyl)-N-(thiazol-2-yl)acetamide(2-9)

To a solution of2-(6-bromo-1-oxoisoindolin-2-yl)-2-(5-fluoro-2-hydroxyphenyl)acetic acid(2-8) in DCM (15 mL) was addedN-(thiazol-2-yl)-1H-imidazole-1-carboxamide (300 mg, 1.70 mmol)(N-(thiazol-2-yl)-1H-imidazole-1-carboxamide was synthesized byfollowing the analogous method which was reported in Angew. Chem. Int.Ed. 2014, 5389-5393). After stirring for overnight, the reaction mixturewas filtered and the resulting filtrate was concentrated under reducedpressure. The residue was purified by flash column chromatography(Hexane/EtOAc=70:30 to 50:50) to afford2-(6-bromo-1-oxoisoindolin-2-yl)-2-(5-fluoro-2-hydroxyphenyl)-N-(thiazol-2-yl)acetamide(2-9) as an off-white solid. (430 mg, 50%)

Step 3.2-(6-Bromo-1-oxoisoindolin-2-yl)-2-(2-((tert-butyldimethylsilyl)oxy)-5-fluorophenyl)-N-(thiazol-2-yl)acetamide(2-10)

To a solution of2-(6-bromo-1-oxoisoindolin-2-yl)-2-(5-fluoro-2-hydroxyphenyl)-N-(thiazol-2-yl)acetamide(2-9) (250 mg, 0.54 mmol) in CH₂Cl₂ (5 mL) was added TBSCl (98 mg, 0.65mmol) and imidazole (55 mg, 0.81 mmol). After stirring for 6 hr, thereaction mixture was diluted with CH₂Cl₂ and washed with water andbrine. The organic layer was dried over sodium sulfate, filtered,concentrated under reduced pressure and purified by columnchromatography on silica gel (DCM:MeOH=10:0 to 8:2) to obtain2-(6-bromo-1-oxoisoindolin-2-yl)-2-(2-((tert-butyldimethylsilyl)oxy)-5-fluorophenyl)-N-(thiazol-2-yl)acetamide(2-10) (296 mg, 95%).

Step 4. tert-Butyl4-(4-(2-(1-(5-fluoro-2-hydroxyphenyl)-2-oxo-2-(thiazol-2-ylamino)ethyl)-3-oxoisoindolin-5-yl)phenyl)piperazine-1-carboxylate(2-11)

A mixture of2-(6-bromo-1-oxoisoindolin-2-yl)-2-(2-((tert-butyldimethylsilyl)oxy)-5-fluorophenyl)-N-(thiazol-2-yl)acetamide(2-10) (380 mg, 0.66 mmol), tert-butyl4-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)piperazine-1-carboxylate(333 mg, 0.857 mmol) and 2 M sodium carbonate (1.32 mL, 2.64 mmol) indioxane (7 mL) was degassed and heated to 100° C. PdCl₂(dppf)₂ (48 mg,0.066 mmol) and Xphos (47 mg, 0.099 mmol) were then added and theresulting reaction mixture was stirred for 2 hr. The reaction mixturewas then cooled down to room temperature and diluted withdichloromethane. The organic layer was washed with water and brine,dried over sodium sulfate, filtered, and concentrated under reducedpressure. The resulting residue was purified by preparative highperformance liquid chromatography (HPLC) to provide tert-butyl4-(4-(2-(1-(5-fluoro-2-hydroxyphenyl)-2-oxo-2-(thiazol-2-ylamino)ethyl)-3-oxoisoindolin-5-yl)phenyl)piperazine-1-carboxylate(2-11) (42 mg, 10%).

Step 5.2-(5-Fluoro-2-hydroxyphenyl)-2-(1-oxo-6-(4-(piperazin-1-yl)phenyl)isoindolin-2-yl)-N-(thiazol-2-yl)acetamidetrifluoroacetic acid salt (2-12)

To a solution of tert-butyl4-(4-(2-(1-(5-fluoro-2-hydroxyphenyl)-2-oxo-2-(thiazol-2-ylamino)ethyl)-3-oxoisoindolin-5-yl)phenyl)piperazine-1-carboxylate(2-11) in DCM (0.8 mL) was added trifluoroacetic acid (0.2 mL). Theresulting solution was stirred for 2 hr and then concentrated underreduced pressure. The resulting residue was concentrated under reducedpressure to give2-(5-fluoro-2-hydroxyphenyl)-2-(1-oxo-6-(4-(piperazin-1-yl)phenyl)isoindolin-2-yl)-N-(thiazol-2-yl)acetamidetrifluoroacetic acid salt (2-12) which was used without furtherpurification.

Step 6.2-(2-((tert-Butyldimethylsilyl)oxy)-5-fluorophenyl)-2-(1-oxo-6-(4-(piperazin-1-yl)phenyl)isoindolin-2-yl)-N-(thiazol-2-yl)acetamide(2-13)

2-(2-((tert-butyldimethylsilyl)oxy)-5-fluorophenyl)-2-(1-oxo-6-(4-(piperazin-1-yl)phenyl)isoindolin-2-yl)-N-(thiazol-2-yl)acetamidewas synthesized by following the analogous procedure of Example 2, Step3 above (145 mg, 80%).

Example 3: Synthesis of2-(6-(4-(4-(3-(2-(2-(2-((2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)ethoxy)propanoyl)piperazin-1-yl)phenyl)-1-oxoisoindolin-2-yl)-2-phenyl-N-(thiazol-2-yl)acetamide(I-1)

Step 1: tert-Butyl3-(2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)ethoxy)propanoate(2-16)

A solution of 2-14 (280 mg, 1.01 mmol), tert-butyl3-(2-(2-(2-aminoethoxy)ethoxy) ethoxy)propanoate (2-15 where n=3, 337mg, 1.22 mmol) and DIEA (0.7 mL, 4.04 mmol) in DMF (5 mL) was stirred at80° C. for overnight. The resulting mixture was diluted with EtOAc andwashed with water five times. The organic layer was dried over Na₂SO₄,filtered, and concentrated under reduced pressure. The resulting residuewas purified by flash column chromatography (DCM/EtOAc=90:10 to 50:50)to afford 2-16 where n=3.

Step 2:3-(2-(2-(2-((2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)ethoxy)propanoic acid trifluoroacetic acid salt (2-17)

To a solution of 2-16 (n=3, 100 mg, 0.19 mmol) in DCM (1.6 mL) was addedtrifluoroacetic acid (0.4 mL). The resulting solution was stirred for 1hr and then concentrated under reduced pressure. The resulting residuewas purified by flash column chromatography (DCM/MeOH=100:0 to 80:20) toafford 2-17 where n=3.

Step 3:2-(6-(4-(4-(3-(2-(2-(2-((2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)ethoxy)propanoyl)piperazin-1-yl)phenyl)-1-oxoisoindolin-2-yl)-2-phenyl-N-(thiazol-2-yl)acetamide(I-1)

To a solution of 2-17 (n=3, 50 mg, 0.08 mmol) and 2-6 (56 mg, 0.11 mmol)in DMF (1 mL) were added EDCI (49 mg, 0.25 mmol) and DIEA (59 μL, 0.34mmol), and the resulting mixture was stirred for 8 hr. The reactionmixture was then diluted with DMSO and purified by preparative HPLC(Method A) to provide desired product I-1 (28 mg, 32%) as a yellowsolid. ¹H NMR 500 MHz (DMSO-d₆) δ 12.71 (s, 1H), 11.09 (s, 1H), 7.88 (s,1H), 7.86 (d, J=7.9 Hz, 1H), 7.65-7.54 (m, 4H), 7.52-7.36 (m, 6H), 7.29(d, J=3.4 Hz, 1H), 7.12 (d, J=8.5 Hz, 1H), 7.08-6.99 (m, 3H), 6.61-6.56(m, 1H), 6.34 (s, 1H), 5.06 (dd, J=5.8, 12.8 Hz, 1H), 4.78 (d, J=17.4Hz, 1H), 4.00 (d, J=17.4 Hz, 1H), 3.70-3.35 (m, 18H), 3.27-3.13 (m, 4H),2.93-2.82 (m, 1H), 2.66-2.52 (m, 4H), 2.07-1.96 (m, 1H). MS m/z: 969.03[M+1]⁺.

Example 4: Synthesis of2-(6-(4-(4-(1-((2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)-3,6,9,12,15-pentaoxaoctadecan-18-oyl)piperazin-1-yl)phenyl)-1-oxoisoindolin-2-yl)-2-phenyl-N-(thiazol-2-yl)acetamide(I-2)

Step 1: tert-butyl1-((2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)-3,6,9,12,15-pentaoxaoctadecan-18-oate(2-19)

A solution of 2-14 (280 mg, 1.01 mmol), tert-butyl1-amino-3,6,9,12,15-pentaoxaoctadecan-18-oate (2-18 where n=5, 337 mg,1.22 mmol) and DIEA (0.7 mL, 4.04 mmol) in DMF (5 mL) was stirred at 80°C. for overnight. The resulting mixture was diluted with EtOAc andwashed with water five times. The organic layer was dried over Na₂SO₄,filtered, and concentrated under reduced pressure. The resulting residuewas purified by flash column chromatography (DCM/EtOAc=90:10 to 50:50)to give 2-19 where n=5.

Step 2:1-((2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)-3,6,9,12,15-pentaoxaoctadecan-18-oicacid trifluoroacetic acid salt (2-20)

To a solution of 2-19 (n=5, 100 mg, 0.19 mmol) in DCM (1.6 mL) was addedtrifluoroacetic acid (0.4 mL). The resulting solution was stirred for 1hr and then concentrated under reduced pressure. The resulting residuewas purified by flash column chromatography (DCM/MeOH=100:0 to 80:20) toafford 2-20 where n=5.

Step 3:2-(6-(4-(4-(1-((2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)-3,6,9,12,15-pentaoxaoctadecan-18-oyl)piperazin-1-yl)phenyl)-1-oxoisoindolin-2-yl)-2-phenyl-N-(thiazol-2-yl)acetamide(I-2)

To a solution of 2-20 (50 mg, 0.08 mmol) and 2-6 (56 mg, 0.11 mmol) inDMF (1 mL) were added EDCI (49 mg, 0.25 mmol) and DIEA (59 μL, 0.34mmol), and the resulting mixture was stirred for 8 hr. The reactionmixture was then diluted with DMSO and purified by preparative HPLC(Method A) to provide desired product I-2 (12 mg, 28%). ¹H NMR 500 MHz(DMSO-d₆) δ 12.70 (s, 1H), 11.09 (s, 1H), 7.88 (s, 1H), 7.85 (d, J=7.6Hz, 1H), 7.69-7.53 (m, 4H), 7.52-7.34 (m, 6H), 7.28 (s, 1H), 7.13 (d,J=8.2 Hz, 1H), 7.08-7.00 (m, 3H), 6.62-6.56 (m, 1H), 6.32 (s, 1H),5.09-5.02 (m, 1H), 4.77 (d, J=17.7 Hz, 1H), 3.99 (d, J=17.4 Hz, 1H),3.74-3.27 (m, 26H), 3.25-3.11 (m, 4H), 2.93-2.81 (m, 1H), 2.67-2.50 (m,4H), 2.07-1.96 (m, 1H). MS m/z: 1057.05 [M+1]⁺.

Example 5: Synthesis of2-(6-(4-(4-(3-(2-(2-(2-((2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)ethoxy)propanoyl)piperazin-1-yl)phenyl)-1-oxoisoindolin-2-yl)-2-(5-fluoro-2-hydroxyphenyl)-N-(thiazol-2-yl)acetamide(I-3)

Step 1:2-(2-((tert-Butyldimethylsilyl)oxy)-5-fluorophenyl)-2-(6-(4-(4-(3-(2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)ethoxy)propanoyl)piperazin-1-yl)phenyl)-1-oxoisoindolin-2-yl)-N-(thiazol-2-yl)acetamide(2-21)

To a solution of 2-17 (n=3, 158 mg, 0.33 mmol) and 2-13 (180 mg, 0.28mmol) in DMF (2 mL) were added HATU (160 mg, 0.414 mmol) and DIEA (0.19mL, 1.10 mmol) at 0° C. The resulting mixture was stirred at roomtemperature for 6 hr. The reaction mixture was diluted with EtOAc andwashed with water and brine. The organic layer was dried over Na₂SO₄,filtered, and concentrated under reduced pressure. The crude product2-21 was used to next step without further purification.

Step 2:2-(6-(4-(4-(3-(2-(2-(2-((2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)ethoxy)propanoyl)piperazin-1-yl)phenyl)-1-oxoisoindolin-2-yl)-2-(5-fluoro-2-hydroxyphenyl)-N-(thiazol-2-yl)acetamide(I-3)

To a solution of crude 2-21 in THE (1 mL) was added 1 M TBAF in THE (1mL). After being stirred for 1 hr, the resulting mixture was dilutedwith DMSO and purified by preparative HPLC (Method A) to afford desiredproduct I-3 (28 mg, 10% as two steps) as a yellow solid. ¹H NMR 500 MHz(DMSO-d₆) δ 12.6 (s, 1H), 11.09 (s, 1H), 9.95 (s, 1H), 7.86 (s, 1H),7.85 (d, J=8.2 Hz, 1H), 7.65-7.52 (m, 4H), 7.49 (d, J=3.4 Hz, 1H), 7.27(d, J=3.4 Hz, 1H), 7.14-7.09 (m, 2H), 7.07-7.00 (m, 3H), 6.91 (dd,J=4.9, 8.9 Hz, 1H), 6.89 (dd, J=3.1, 9.2 Hz, 1H), 6.58 (t, J=5.5 Hz,1H), 6.33 (s, 1H), 5.05 (dd, J=5.5, 12.8 Hz, 1H), 4.63 (d, J=17.4 Hz,1H), 4.00 (d, J=17.3 Hz, 1H), 3.67-3.40 (m, 18H), 3.25-3.12 (m, 4H),2.93-2.83 (m, 1H), 2.66-2.50 (m, 4H), 2.07-1.95 (m, 1H). MS m/z: 1002.96[M+1]⁺.

Example 6: Synthesis of2-(6-(4-(4-(17-((2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)-2,16-dioxo-6,9,12-trioxa-3,15-diazaheptadecyl)piperazin-1-yl)phenyl)-1-oxoisoindolin-2-yl)-2-phenyl-N-(thiazol-2-yl)acetamide(I-4)

Step 1: ter-Butyl2-(4-(4-(3-oxo-2-(2-oxo-1-phenyl-2-(thiazol-2-ylamino)ethyl)isoindolin-5-yl)phenyl)piperazin-1-yl)acetate(2-23)

A solution of 2-6 (300 mg, 0.59 mmol), tert-butyl bromoacetate (2-22,103 μL, 0.71 mmol), and K₂CO₃ (244 mg, 1.77 mmol) in DMF (2.5 mL) wasstirred at 80° C. for 8 hr. The resulting mixture was then diluted withEtOAc and washed four times with water. The organic layer was dried overNa₂SO₄, filtered, and concentrated under reduced pressure. The residuewas purified by flash column chromatography (DCM/1M NH₃ in MeOH=100:0 to80:20) to give product 2-23.

Step 2:2-(4-(4-(3-Oxo-2-(2-oxo-1-phenyl-2-(thiazol-2-ylamino)ethyl)isoindolin-5-yl)phenyl)piperazin-1-yl)aceticacid (2-24)

To a solution of the 2-23 in DCM (4 mL) was added TFA (1 mL) and theresulting mixture was stirred at room temperature. Upon reactioncompletion, the fraction mixture was concentrated and the crude 2-24 wasused to next step without further purification.

Step 3:2-(6-(4-(4-(17-((2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)-2,16-dioxo-6,9,12-trioxa-3,15-diazaheptadecyl)piperazin-1-yl)phenyl)-1-oxoisoindolin-2-yl)-2-phenyl-N-(thiazol-2-yl)acetamide(I-4)

To a solution of 2-24 (57 mg, 0.10 mmol) and 2-25 (50 mg, 0.10 mmol) inDMF (1 mL) was added HATU (76 mg, 0.20 mmol), DIEA (122 μL, 0.70 mmol),and the resulting mixture was stirred for 3 hr. The reaction mixture wasthen diluted with DMSO and purified by preparative HPLC to give desiredproduct I-4 (34 mg, 32%) as an off-white solid. ¹H NMR 500 MHz (DMSO-d₆)δ 12.70 (s, 1H), 11.12 (s, 1H), 10.33-9.95 (br, 1H), 8.77-8.43 (br, 1H),8.04-7.97 (m, 1H), 7.90 (s, 1H), 7.86 (d, J=7.9 Hz, 1H), 7.81 (t, J=7.9Hz, 1H), 7.65 (d, J=5.8 Hz, 2H), 7.53-7.36 (m, 8H), 7.28 (d, J=3.1 Hz,1H), 7.08 (d, J=8.2 Hz, 2H), 6.32 (s, 1H), 5.11 (dd, J=5.2, 12.5 Hz,1H), 4.81-4.74 (m, 3H), 4.00 (d, J=17.4 Hz, 1H), 3.52 (s, 8H), 3.48-3.44(m, 4H), 3.44-3.29 (m, 14H), 2.95-2.84 (m, 1H), 2.65-2.50 (m, 2H),2.08-2.00 (m, 1H). MS m/z: 1055.97 [M+1]⁺

Example 7:2-(6-(4-(4-(2-((4-(2-((2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamido)butyl)amino)-2-oxoethyl)piperazin-1-yl)phenyl)-1-oxoisoindolin-2-yl)-2-phenyl-N-(thiazol-2-yl)acetamide(I-5)

To a solution of 2-24 (57 mg, 0.10 mmol) and 2-26 (50 mg, 0.10 mmol) inDMF (1 mL) was added HATU (76 mg, 0.20 mmol), DIEA (122 μL, 0.70 mmol),and the resulting mixture was stirred for 3 hr. The reaction mixture wasthen diluted with DMSO and purified by preparative HPLC to give desiredproduct I-5 (14 mg, 28%) as an off-white solid. ¹H NMR 500 MHz (DMSO-d₆)δ 12.71 (s, 1H), 11.12 (s, 1H), 10.39-10.12 (br, 1H), 8.69-8.48 (br,1H), 8.06-8.01 (m, 1H), 7.91 (s, 1H), 7.88 (d, J=7.9 Hz, 1H), 7.83 (t,J=7.9 Hz, 1H), 7.66 (d, J=8.5 Hz, 2H), 7.62 (d, J=8.5 Hz, 1H), 7.54-7.37(m, 7H), 7.29 (d, J=3.7 Hz, 1H), 7.09 (d, J=8.9 Hz, 2H), 6.33 (s, 1H),5.13 (dd, J=5.5, 13.1 Hz, 1H), 4.82-4.75 (m, 3H), 4.01 (d, J=17.7 Hz,1H), 3.42-3.29 (s, 10H), 3.21-3.12 (m, 4H), 2.95-2.85 (m, 1H), 2.66-2.50(m, 2H), 2.08-2.00 (m, 1H), 1.47 (br, 4H). MS m/z: 951.96 [M+1]⁺.

Example 8.(2S,4R)-1-((2S)-3,3-Dimethyl-2-(2-(4-(4-(3-oxo-2-(2-oxo-1-phenyl-2-(thiazol-2-ylamino)ethyl)isoindolin-5-yl)phenyl)piperazin-1-yl)acetamido)butanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide(I-6)

To a solution of 2-24 (1 equiv, based on tert-butyl ester) and 2-27 (24mg, 0.055 mmol) in CH₂Cl₂ (1 mL) was added EDCI (21 mg, 0.11 mmol), DIEA(38 μL, 0.22 mmol), and the resulting mixture was stirred for 6 hr. Thereaction mixture was concentrated under reduced pressure then dilutedwith DMSO and purified by preparative HPLC to give desired product I-6(21 mg, 40%) as an off-white solid. MS m/z: 980.64 [M+1]⁺.

Example 9. Alternative synthesis of2-(6-(4-(4-(3-(2-(2-(2-((2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)ethoxy)propanoyl)piperazin-1-yl)phenyl)-1-oxoisoindolin-2-yl)-2-(5-fluoro-2-hydroxyphenyl)-N-(thiazol-2-yl)acetamide(I-3)

Step 1:2-(6-(4-(4-(3-(2-((2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)propanoyl)piperazin-1-yl)phenyl)-1-oxoisoindolin-2-yl)-2-(5-fluoro-2-(methoxymethoxy)phenyl)-N-(thiazol-2-yl)acetamide(2-29)

To a solution of 2-17 (158 mg, 0.33 mmol) and 2-28 (180 mg, 0.28 mmol)in DMF (2 mL) was added HATU (160 mg, 0.414 mmol) and DIEA (0.19 mL,1.10 mmol) at 0° C. and the resulting mixture was stirred at roomtemperature for 6 hours. The reaction mixture was diluted with EtOAc andwashed with water and brine. The organic layer was dried over Na₂SO₄,filtered, and concentrated under reduced pressure. The residue wascarried forward in the next step without further purification.

Step 2:2-(6-(4-(4-(3-(2-(2-(2-((2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)ethoxy)propanoyl)piperazin-1-yl)phenyl)-1-oxoisoindolin-2-yl)-2-(5-fluoro-2-hydroxyphenyl)-N-(thiazol-2-yl)acetamide(1-3)

To a solution of crude 2-29 in THE (1 mL) was added 1 M TBAF in THE (1mL). After stirring for 1 hour, the resulting mixture was diluted withDMSO and purified by preparative HPLC to afford desired product (28 mg,10% as two steps) as a yellow solid. MS m/z: 1002.96 [M+1]⁺; ¹H NMR 500MHz (DMSO-d₆) δ 12.6 (s, 1H), 11.09 (s, 1H), 9.95 (s, 1H), 7.86 (s, 1H),7.85 (d, J=8.2 Hz, 1H), 7.65-7.52 (m, 4H), 7.49 (d, J=3.4 Hz, 1H), 7.27(d, J=3.4 Hz, 1H), 7.14-7.09 (m, 2H), 7.07-7.00 (m, 3H), 6.91 (dd,J=4.9, 8.9 Hz, 1H), 6.89 (dd, J=3.1, 9.2 Hz, 1H), 6.58 (t, J=5.5 Hz,1H), 6.33 (s, 1H), 5.05 (dd, J=5.5, 12.8 Hz, 1H), 4.63 (d, J=17.4 Hz,1H), 4.00 (d, J=17.3 Hz, 1H), 3.67-3.40 (m, 18H), 3.25-3.12 (m, 4H),2.93-2.83 (m, 1H), 2.66-2.50 (m, 4H), 2.07-1.95 (m, 1H).

Example 10: Synthesis of2-(6-(4-(4-(3-(2-(2-((2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)propanoyl)piperazin-1-yl)phenyl)-1-oxoisoindolin-2-yl)-2-phenyl-N-(thiazol-2-yl)acetamide(I-7)

Compound 1-7 was prepared according to the synthesis of Compound I-1 asshown in Example 3. MS m/z: 925.78 [M+1]⁺; ¹H NMR (500 MHz, DMSO-d₆)δ=12.70 (br s, 1H), 11.09 (br s, 1H), 7.88 (s, 1H), 7.85 (d, J=8.2 Hz,1H), 7.66-7.54 (m, 4H), 7.52-7.36 (m, 6H), 7.31-7.25 (m, 1H), 7.13 (d,J=8.9 Hz, 1H), 7.03 (d, J=7.3 Hz, 3H), 6.64-6.55 (m, 1H), 6.33 (s, 1H),5.05 (dd, J=5.3, 13.0 Hz, 1H), 4.77 (d, J=17.4 Hz, 1H), 4.00 (d, J=17.4Hz, 1H), 3.71-3.48 (m, 12H), 3.48-3.40 (m, 2H), 3.25-3.09 (m, 4H),2.92-2.81 (m, 1H), 2.63-2.51 (m, 4H), 2.07-1.96 (m, 1H).

Example 11: Synthesis of2-(6-(4-(4-(3-(2-((2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)propanoyl)piperazin-1-yl)phenyl)-1-oxoisoindolin-2-yl)-2-phenyl-N-(thiazol-2-yl)acetamide(I-8)

Compound 1-8 was prepared according to the synthesis of Compound I-1 asshown in Example 3. MS m/z: 881.89 [M+1]⁺; ¹H NMR (500 MHz, DMSO-d₆)δ=12.70 (br s, 1H), 11.10 (s, 1H), 7.88 (s, 1H), 7.87-7.83 (m, 1H),7.62-7.55 (m, 4H), 7.52-7.35 (m, 6H), 7.28 (d, J=3.4 Hz, 1H), 7.13 (d,J=8.9 Hz, 1H), 7.01 (t, J=8.1 Hz, 3H), 6.58 (t, J=5.0 Hz, 1H), 6.33 (s,1H), 5.05 (dd, J=5.5, 12.8 Hz, 1H), 4.77 (d, J=17.4 Hz, 1H), 4.00 (d,J=17.7 Hz, 1H), 3.71 (t, J=6.4 Hz, 2H), 3.64-3.59 (m, 6H), 3.48-3.44 (m,2H), 3.20-3.13 (m, 4H), 2.90-2.82 (m, 1H), 2.64 (t, J=6.4 Hz, 2H),2.59-2.52 (m, 2H), 2.03-1.97 (m, 1H).

Example 12: Synthesis of2-(6-(4-(4-(1-((2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)-3,6,9,12-tetraoxapentadecan-15-oyl)piperazin-1-yl)phenyl)-1-oxoisoindolin-2-yl)-2-phenyl-N-(thiazol-2-yl)acetamide(I-9)

Compound 1-9 was prepared according to the synthesis of Compound I-1 asshown in Example 3. MS m/z: 1013.91 [M+1]⁺; ¹H NMR (500 MHz, DMSO-d₆)δ=12.70 (br s, 1H), 11.09 (s, 1H), 7.88 (s, 1H), 7.85 (dd, J=1.5, 7.9Hz, 1H), 7.63-7.54 (m, 4H), 7.50-7.37 (m, 6H), 7.28 (d, J=3.4 Hz, 1H),7.12 (d, J=8.5 Hz, 1H), 7.06-7.01 (m, 3H), 6.59 (t, J=5.8 Hz, 1H), 6.33(s, 1H), 5.05 (dd, J=5.5, 12.8 Hz, 1H), 4.77 (d, J=17.7 Hz, 1H), 3.99(d, J=17.7 Hz, 1H), 3.65-3.59 (m, 8H), 3.56-3.53 (m, 2H), 3.52-3.43 (m,12H), 3.24-3.14 (m, 4H), 2.88 (ddd, J=5.5, 13.8, 17.0 Hz, 1H), 2.64-2.52(m, 4H), 2.04-1.99 (m, 1H).

Example 13: Synthesis of2-(6-(4-(4-(2-(2-(2-(2-((2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)ethoxy)ethyl)piperazin-1-yl)phenyl)-1-oxoisoindolin-2-yl)-2-(5-fluoro-2-hydroxyphenyl)-N-(thiazol-2-yl)acetamide(I-10)

Step 1:2-(2,6-Dioxopiperidin-3-yl)-4-((2-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)ethyl)amino)isoindoline-1,3-dione(2-37)

To a solution of 2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethan-1-ol (244mg, 1.64 mmol) in DMF (5 mL) was added DIEA (0.28 mL, 2.18 mmol). Afterstirring at 80° C. for overnight, the reaction mixture was cooled toroom temperature and concentrated under reduced pressure. The residuewas purified by flash column chromatography (DCM:MeOH=100:0 to 80:20) toafford 2-37 (284 mg, 58%) as a dark green liquid.

Step 2:2-(2-(2-(2-((2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)ethoxy)ethylmethanesulfonate (2-38)

To a solution of 2-37 (225 mg, 0.50 mmol) in dry DCM was added DIEA (131μL, 0.75 mmol) and MsCl (46 μL, 0.60 mmol) at 0° C. The mixture wasgradually warmed up to room temperature for 1 hour. After completion,the reaction mixture was concentrated and purified by flash columnchromatography (hexane:EtOAc=50:50 to DCM:EtOAc 50:50) to afford 2-38(169 mg, 64%) as a yellow sticky oil.

Step 3:2-(6-(4-(4-(2-(2-((2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethyl)piperazin-1-yl)phenyl)-1-oxoisoindolin-2-yl)-2-(5-fluoro-2-(methoxymethoxy)phenyl)-N-(thiazol-2-yl)acetamide(2-40)

To a solution of 2-38 (54 mg, 0.102 mmol) and 2-39 (60 mg, 0.102 mmol)in DMF (2 mL) was added DIEA (53 μL, 0.307 mmol). After stirring at 80°C. for 4 hours, the reaction mixture was diluted with DMSO and purifiedby preparative HPLC to obtain 2-40 (88 mg, 85%).

Step 4:2-(6-(4-(4-(2-(2-(2-(2-((2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)ethoxy)ethyl)piperazin-1-yl)phenyl)-1-oxoisoindolin-2-yl)-2-(5-fluoro-2-hydroxyphenyl)-N-(thiazol-2-yl)acetamide(I-10)

To a solution of 2-40 (88 mg, 0.086 mmol) in dry DCM (4 mL) was addedTFA (1 mL) at 0° C. After stirring for 5 hours, the reaction mixture wasconcentrated under reduced pressure and purified by flash columnchromatography (DCM:1 N NH₃ in MeOH=100:0 to 80:20) to afford I-10 (27mg, 32%) as a yellowish solid. MS m/z: 975.41 [M+1]⁺; ¹H NMR 500 MHz(DMSO-d₆) δ 12.62 (bs, 1H), 11.10 (s, 1H), 9.96 (s, 1H), 7.86 (s, 1H)7.84 (d, J=8.2 Hz, 1H), 7.62-7.54 (m, 4H), 7.49 (d, J=3.4 Hz, 1H), 7.27(d, J=3.4 Hz, 1H), 7.17-7.09 (m, 2H), 7.06-6.99 (m, 3H), 6.94-6.89 (m,1H), 6.89-6.85 (m, 1H), 6.62 (br t, J=5.6 Hz, 1H), 6.34 (s, 1H), 5.05(dd, J=12.5, 5.3 Hz, 1H), 4.62 (d, J=17.4 Hz, 1H), 4.00 (d, J=17.7 Hz,1H), 3.63 (t, J=5.3 Hz, 2H), 3.60-3.40 (m, 16H), 3.17 (br, 4H),2.92-2.82 (m, 1H), 2.66-2.50 (m, 4H), 2.05-1.96 (m, 1H).

Example 14: Synthesis of2-(6-(4-(4-(6-((2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)hexyl)piperazin-1-yl)phenyl)-1-oxoisoindolin-2-yl)-2-(5-fluoro-2-hydroxyphenyl)-N-(thiazol-2-yl)acetamide(I-11)

Compound I-11 was prepared in a manner similar to the synthesis ofCompound I-10 as shown in Example 13. Step 4 of the synthesis ofCompound 1-11 is shown above in Example 14. MS m/z: 899.70 [M+1]⁺; ¹HNMR 500 MHz (DMSO-d₆) δ 12.58 (bs, 1H), 11.09 (s, 1H), 9.96 (s, 1H),7.88-7.82 (m, 2H), 7.62-7.55 (m, 4H), 7.50-7.47 (m, 1H), 7.27-7.24 (m,1H), 7.14-7.07 (m, 2H), 7.04-6.99 (m, 3H), 6.93-6.88 (m, 1H), 6.88-6.84(m, 1H), 6.53 (br t, J=5.5 Hz, 1H), 6.33 (s, 1H), 5.05 (dd, J=12.6, 5.4Hz, 1H), 4.62 (d, J=17.4 Hz, 1H), 4.00 (d, J=17.4 Hz, 1H), 3.39-3.25 (m,4H), 3.22-3.13 (m, 4H), 2.93-2.83 (m, 1H), 2.64-2.50 (m, 4H), 2.34-2.27(m, 2H), 2.06-1.98 (m, 1H), 1.63-1.55 (m, 2H), 1.52-1.43 (m, 2H),1.42-1.28 (m, 4H).

Example 15: Synthesis of2-(6-(4-(4-(8-((2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)octyl)piperazin-1-yl)phenyl)-1-oxoisoindolin-2-yl)-2-(5-fluoro-2-hydroxyphenyl)-N-(thiazol-2-yl)acetamide(1-12)

Compound I-12 was prepared in a manner similar to the synthesis ofCompound I-10 as shown in Example 13. Step 4 of the synthesis ofCompound I-12 is shown above in Example 15. MS m/z: 899.70 [M+1]⁺; ¹HNM/R 500 MHz (DMSO-d₆) δ 12.58 (bs, 1H), 11.09 (s, 1H), 9.96 (s, 1H),7.88-7.82 (m, 2H), 7.62-7.55 (m, 4H), 7.50-7.47 (m, 1H), 7.27-7.24 (m,1H), 7.14-7.07 (m, 2H), 7.04-6.99 (m, 3H), 6.93-6.88 (m, 1H), 6.88-6.84(m, 1H), 6.53 (br t, J=5.5 Hz, 1H), 6.33 (s, 1H), 5.05 (dd, J=12.6, 5.4Hz, 1H), 4.62 (d, J=17.4 Hz, 1H), 4.00 (d, J=17.4 Hz, 1H), 3.39-3.25 (m,4H), 3.22-3.13 (m, 4H), 2.93-2.83 (m, 1H), 2.64-2.50 (m, 4H), 2.34-2.27(m, 2H), 2.06-1.98 (m, 1H), 1.63-1.55 (m, 2H), 1.52-1.43 (m, 2H),1.42-1.28 (in, 4H).

Example 16: Synthesis of2-(6-(4-(4-(8-((2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)octyl)piperazin-1-yl)phenyl)-1-oxoisoindolin-2-yl)-2-(5-fluoro-2-hydroxyphenyl)-N-(thiazol-2-yl)acetamide(I-13)

Compound I-13 was prepared in a manner similar to the synthesis ofCompound I-10 as shown in Example 13. Step 4 of the synthesis ofCompound 1-13 is shown in Example 16. MS m/z: 1019.50 [M+1]⁺.

Example 17: Synthesis of2-(6-(6-(4-(2-(2-(2-(2-((2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)ethoxy)ethyl)piperazin-1-yl)pyridin-3-yl)-1-oxoisoindolin-2-yl)-2-(2-hydroxyphenyl)-N-(thiazol-2-yl)acetamide(I-14)

Compound I-14 was prepared according to the synthesis of Compound I-10as shown in Example 13. Step 4 of the synthesis of Compound I-14 isshown in Example 17. MS m/z: 959.09 [M+1]⁺.

Example 18: Synthesis of2-(6-(4-(4-(2-(2-(2-(3-((2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)azetidin-1-yl)ethoxy)ethoxy)ethyl)piperazin-1-yl)phenyl)-1-oxoisoindolin-2-yl)-2-phenyl-N-(thiazol-2-yl)acetamide(I-15)

Compound I-15 was prepared in a manner similar to the synthesis ofCompound I-10 as shown in Example 13. MS m/z: 952.54 [M+1]⁺; ¹H NMR 500MHz (DMSO-d₆) δ 12.31 (bs s, 1H), 11.10 (s, 1H), 7.87-7.85 (m, 1H) 7.83(dd, J=7.9, 1.8 Hz, 1H), 7.60-7.53 (m, 4H), 7.51-7.37 (m, 6H), 7.28 (d,J=3.7 Hz, 1H), 7.06 (d, J=7.0 Hz, 1H), 7.04-7.00 (m, 1H), 6.99 (d, J=8.5Hz, 2H), 6.59 (d, J=6.7 Hz, 1H), 6.32 (s, 1H), 5.05 (dd, J=12.8, 5.5 Hz,1H), 4.77 (d, J=17.4 Hz, 1H), 4.27-4.20 (m, 1H), 3.99 (d, J=17.7 Hz,1H), 3.69 (t, J=7.0 Hz, 2H), 3.55 (t, J=5.8 Hz, 2H), 3.53-3.42 (m, 4H),3.40 (t, J=5.8 Hz, 2H), 3.22-3.15 (m, 4H), 2.97 (t, J=6.7 Hz, 2H),2.93-2.83 (m, 1H), 2.65-2.47 (m, 10H), 2.04-1.96 (m, 1H).

Example 19: Synthesis of2-(6-(4-(4-(2-(2-(2-(2-(3-((2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)azetidin-1-yl)ethoxy)ethoxy)ethoxy)ethyl)piperazin-1-yl)phenyl)-1-oxoisoindolin-2-yl)-2-phenyl-N-(thiazol-2-yl)acetamide(I-16)

Compound I-16 was prepared according to the synthesis of Compound I-10as shown in Example 13. MS m/z: 996.80 [M+1]⁺; ¹H NMR 500 MHz (DMSO-d₆)δ 12.50 (bs s, 1H), 11.10 (s, 1H), 7.86 (s, 1H) 7.83 (dd, J=7.9, 1.5 Hz,1H), 7.62-7.53 (m, 4H), 7.52-7.36 (m, 6H), 7.27 (d, J=3.4 Hz, 1H), 7.06(d, J=7.0 Hz, 1H), 7.04-6.93 (m, 3H), 6.58 (d, J=7.0 Hz, 1H), 6.32 (s,1H), 5.05 (dd, J=12.8, 5.5 Hz, 1H), 4.76 (d, J=17.4 Hz, 1H), 4.27-4.16(m, 1H), 3.98 (d, J=17.7 Hz, 1H), 3.67 (t, J=7.0 Hz, 2H), 3.58-3.43 (m,10H), 3.38 (t, J=5.8 Hz, 2H), 3.21-3.10 (m, 4H), 2.97 (t, J=6.7 Hz, 2H),2.92-2.80 (m, 1H), 2.73-2.45 (m, 10H), 2.07-1.95 (m, 1H).

Example 20: Synthesis of2-(6-(4-(4-(6-((2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)hexyl)piperazin-1-yl)phenyl)-1-oxoisoindolin-2-yl)-2-phenyl-N-(thiazol-2-yl)acetamide(I-17)

Compound I-17 was prepared in a manner similar to the synthesis ofCompound I-10 as shown in Example 13. MS m/z: 865.92 [M+1]⁺; ¹H NMR (500MHz, DMSO-d₆) δ=12.70 (br s, 1H), 11.09 (s, 1H), 7.88 (s, 1H), 7.85 (d,J=7.9 Hz, 1H), 7.66-7.54 (m, 4H), 7.50-7.37 (m, 6H), 7.28 (d, J=3.1 Hz,1H), 7.10 (d, J=8.5 Hz, 1H), 7.02 (d, J=6.7 Hz, 3H), 6.56-6.51 (m, 1H),6.32 (s, 1H), 5.05 (dd, J=5.5, 12.8 Hz, 1H), 4.77 (d, J=17.7 Hz, 1H),3.99 (d, J=17.4 Hz, 1H), 3.31-3.28 (m, 3H), 3.26-3.10 (m, 4H), 2.92-2.84(m, 1H), 2.65-2.52 (m, 4H), 2.42-2.16 (m, 3H), 2.05-1.99 (m, 1H),1.63-1.56 (m, 2H), 1.53-1.45 (m, 2H), 1.41-1.32 (m, 4H).

Example 21: Synthesis of2-(6-(4-(4-(2-(2-(2-(2-((2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)ethoxy)ethyl)piperazin-1-yl)phenyl)-1-oxoisoindolin-2-yl)-2-phenyl-N-(thiazol-2-yl)acetamide(I-18)

Compound I-18 was prepared in a manner similar to the synthesis ofCompound I-10 as shown in Example 13. MS m/z: 941.90 [M+1]⁺; ¹H NMR (500MHz, DMSO-d₆) δ=12.67 (br s, 1H), 11.10 (br s, 1H), 7.87 (s, 1H), 7.84(d, J=8.5 Hz, 1H), 7.63-7.54 (m, 4H), 7.50-7.36 (m, 6H), 7.26 (br s,1H), 7.14 (d, J=8.5 Hz, 1H), 7.02 (dd, J=7.9, 15.9 Hz, 3H), 6.61 (t,J=5.6 Hz, 1H), 6.31 (s, 1H), 5.05 (dd, J=5.3, 12.7 Hz, 1H), 4.78 (d,J=17.1 Hz, 1H), 3.99 (d, J=17.4 Hz, 1H), 3.65-3.61 (m, 2H), 3.58-3.45(m, 12H), 3.21-3.12 (m, 4H), 2.92-2.83 (m, 1H), 2.65-2.51 (m, 8H),2.04-1.99 (m, 1H).

Example 22: Synthesis of2-(6-(4-(4-(8-((2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)octyl)piperazin-1-yl)phenyl)-1-oxoisoindolin-2-yl)-2-phenyl-N-(thiazol-2-yl)acetamide(I-19)

Compound I-19 was prepared in a manner similar to the synthesis ofCompound I-10 as shown in Example 13. MS m/z: 893.72 [M+1]⁺; ¹H NMR (500MHz, DMSO-d₆) δ=12.70 (br s, 1H), 11.09 (s, 1H), 7.88 (s, 1H), 7.85 (d,J=7.9 Hz, 1H), 7.62-7.55 (m, 4H), 7.50-7.37 (m, 6H), 7.28 (d, J=3.4 Hz,1H), 7.09 (d, J=8.5 Hz, 1H), 7.04-7.00 (m, 3H), 6.52 (t, J=5.6 Hz, 1H),6.32 (s, 1H), 5.05 (dd, J=5.3, 12.7 Hz, 1H), 4.77 (d, J=17.7 Hz, 1H),3.99 (d, J=17.4 Hz, 1H), 3.31-3.27 (m, 3H), 3.24-3.11 (m, 4H), 2.92-2.84(m, 1H), 2.64-2.52 (m, 4H), 2.43-2.21 (m, 3H), 2.05-1.99 (m, 1H),1.61-1.55 (m, 2H), 1.51-1.43 (m, 2H), 1.38-1.28 (m, 8H).

Example 23: Synthesis of2-(6-(4-(4-(14-((2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)-3,6,9,12-tetraoxatetradecyl)piperazin-1-yl)phenyl)-1-oxoisoindolin-2-yl)-2-phenyl-N-(thiazol-2-yl)acetamide(I-20)

Compound I-20 was prepared in a manner similar to the synthesis ofCompound I-10 as shown in Example 13. MS m/z: 985.93 [M+1]⁺; ¹H NMR (500MHz, DMSO-d₆) δ=12.70 (br s, 1H), 11.10 (s, 1H), 7.87 (d, J=0.9 Hz, 1H),7.84 (dd, J=1.7, 8.1 Hz, 1H), 7.60-7.55 (m, 4H), 7.50-7.37 (m, 6H), 7.28(d, J=3.7 Hz, 1H), 7.13 (d, J=8.5 Hz, 1H), 7.04-6.99 (m, 3H), 6.59 (t,J=5.8 Hz, 1H), 6.32 (s, 1H), 5.05 (dd, J=5.5, 12.8 Hz, 1H), 4.77 (d,J=17.7 Hz, 1H), 3.99 (d, J=17.7 Hz, 1H), 3.63-3.59 (m, 2H), 3.58-3.49(m, 14H), 3.46 (q, J=5.7 Hz, 2H), 3.21-3.14 (m, 4H), 2.92-2.84 (m, 1H),2.65-2.51 (m, 8H), 2.04-1.98 (m, 1H).

Example 24: Synthesis of2-(6-(4-(4-(2-(2-(2-((2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)ethyl)piperazin-1-yl)phenyl)-1-oxoisoindolin-2-yl)-2-phenyl-N-(thiazol-2-yl)acetamide(I-21)

Compound I-21 was prepared in a manner similar to the synthesis ofCompound I-10 as shown in Example 13. MS m/z: 897.95 [M+1]⁺; ¹H NMR (500MHz, DMSO-d₆) δ=12.70 (br s, 1H), 11.10 (s, 1H), 7.89 (s, 1H), 7.88-7.84(m, 1H), 7.66-7.55 (m, 4H), 7.51-7.36 (m, 6H), 7.28 (d, J=3.7 Hz, 1H),7.15 (d, J=8.5 Hz, 1H), 7.05 (d, J=7.3 Hz, 3H), 6.60 (t, J=5.6 Hz, 1H),6.32 (s, 1H), 5.05 (dd, J=5.3, 12.7 Hz, 1H), 4.78 (d, J=17.7 Hz, 1H),4.00 (d, J=17.7 Hz, 1H), 3.80-3.76 (m, 2H), 3.68-3.57 (m, 8H), 3.51-3.47(m, 4H), 3.21-3.06 (m, 4H), 2.90-2.80 (m, 1H), 2.61-2.50 (m, 4H),2.04-1.97 (m, 1H).

Example 25: Synthesis of2-(6-(6-(4-(2-(2-(2-(2-((2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)ethoxy)ethyl)piperazin-1-yl)pyridin-3-yl)-1-oxoisoindolin-2-yl)-2-phenyl-N-(thiazol-2-yl)acetamide(I-22)

Compound I-22 was prepared in a manner similar to the synthesis ofCompound I-10 as shown in Example 13. MS m/z: 942.95 [M+1]⁺; ¹H NMR (500MHz, DMSO-d₆) δ=12.70 (s, 1H), 11.10 (s, 1H), 8.49 (br s, 1H), 7.99-7.91(m, 1H), 7.90 (s, 1H), 7.86 (dd, J=1.7, 8.1 Hz, 1H), 7.63-7.55 (m, 2H),7.50-7.37 (m, 6H), 7.28 (d, J=3.7 Hz, 1H), 7.14 (d, J=8.5 Hz, 1H), 7.03(d, J=7.0 Hz, 1H), 6.92 (br s, 1H), 6.60 (t, J=5.8 Hz, 1H), 6.33 (s,1H), 5.05 (dd, J=5.3, 12.7 Hz, 1H), 4.78 (d, J=17.7 Hz, 1H), 4.00 (d,J=17.7 Hz, 1H), 3.69-3.40 (m, 18H), 2.88 (ddd, J=5.3, 13.9, 17.1 Hz,1H), 2.66-2.50 (m, 8H), 2.05-1.98 (m, 1H).

Example 26: Synthesis of(2S,4R)-1-((14S)-14-(tert-Butyl)-12-oxo-1-(4-(5-(3-oxo-2-(2-oxo-1-phenyl-2-(thiazol-2-ylamino)ethyl)isoindolin-5-yl)pyridin-2-yl)piperazin-1-yl)-3,6,9-trioxa-13-azapentadecan-15-oyl)-4-hydroxy-N—((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide(I-23)

Step 1: tert-Butyl3-(2-(4-(5-(3-oxo-2-(2-oxo-1-phenyl-2-(thiazol-2-ylamino)ethyl)isoindolin-5-yl)pyridin-2-yl)piperazin-1-yl)ethoxy)propanoate(2-70)

To a solution of 2-48A (204 mg, 0.40 mmol) in DMF (1.5 mL) was addedtert-butyl 3-(2-(2-(2-bromoethoxy)ethoxy)ethoxy)propanoate (2-69, 136mg, 0.40 mmol) and DIEA (0.14 mL, 0.80 mmol). The resulting mixture wasstirred at 80° C. for 8 hours. After cooling to room temperature, thereaction mixture was diluted with EtOAc and washed with water fivetimes. The organic layer was dried over Na₂SO₄, filtered andconcentrated under reduced pressure. The residue was purified by flashcolumn chromatography (hexane:EtOAc=100:0 to 0:100 then, DCM:MeOH=100:0to 80:20) to afford 2-70 (191 mg, 62%) as an off-white solid.

Step 2:(2S,4R)-1-((14S)-14-(tert-Butyl)-12-oxo-1-(4-(5-(3-oxo-2-(2-oxo-1-phenyl-2-(thiazol-2-ylamino)ethyl)isoindolin-5-yl)pyridin-2-yl)piperazin-1-yl)-3,6,9-trioxa-13-azapentadecan-15-oyl)-4-hydroxy-N—((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide(I-23)

To a solution of 2-70 (150 mg, 0.19 mmol) in DCM (3.5 mL) was added TFA(1.5 mL). After stirring for 1 hour, the reaction mixture wasconcentrated under reduced pressure and the residue was purified bypreparative HPLC to afford3-(2-(2-(2-(4-(5-(3-oxo-2-(2-oxo-1-phenyl-2-(thiazol-2-ylamino)ethyl)isoindolin-5-yl)pyridin-2-yl)piperazin-1-yl)ethoxy)ethoxy)ethoxy)propanoicacid (114 mg, 82%) as an off-white solid. Then, to a solution of 2-71(31 mg, 0.07 mmol) and3-(2-(2-(2-(4-(5-(3-oxo-2-(2-oxo-1-phenyl-2-(thiazol-2-ylamino)ethyl)isoindolin-5-yl)pyridin-2-yl)piperazin-1-yl)ethoxy)ethoxy)ethoxy)propanoicacid (63 mg, 0.088 mmol) in DMF (1.0 mL) was added EDCI (20 mg, 0.11mmol), HOBt (9 mg, 0.07 mmol) and DIEA (0.05 mL, 0.28 mmol). Afterstirring overnight, the reaction mixture was diluted with DMSO andpurified by preparative HPLC to afford I-23 (34 mg, 43%) as an off-whitesolid. MS m/z: 1141.74 [M+1]⁺; ¹H NMR (500 MHz, DMSO-d₆) δ=12.69 (br s,1H), 8.97 (s, 1H), 8.50 (d, J=2.4 Hz, 1H), 8.37 (d, J=7.6 Hz, 1H),7.94-7.90 (m, 2H), 7.86 (dd, J=1.7, 8.1 Hz, 2H), 7.61 (d, J=7.9 Hz, 1H),7.52-7.31 (m, 10H), 7.28 (d, J=3.4 Hz, 1H), 6.92 (d, J=8.9 Hz, 1H), 6.32(s, 1H), 5.10 (d, J=3.7 Hz, 1H), 4.95-4.87 (m, 1H), 4.78 (d, J=17.4 Hz,1H), 4.53 (d, J=9.5 Hz, 1H), 4.42 (t, J=8.1 Hz, 1H), 4.27 (br s, 1H),4.00 (d, J=17.7 Hz, 1H), 3.65-3.42 (m, 18H), 2.58-2.51 (m, 7H), 2.45 (s,3H), 2.35 (dt, J=6.1, 14.6 Hz, 1H), 2.04-1.97 (m, 1H), 1.79 (ddd, J=4.7,8.4, 12.8 Hz, 1H), 1.37 (d, J=7.0 Hz, 3H), 0.93 (s, 9H).

Example 27: Synthesis of(2S,4R)-1-((2S)-3,3-Dimethyl-2-(3-(2-(2-(4-(5-(3-oxo-2-(2-oxo-1-phenyl-2-(thiazol-2-ylamino)ethyl)isoindolin-5-yl)pyridin-2-yl)piperazin-1-yl)ethoxy)ethoxy)propanamido)butanoyl)-4-hydroxy-N—((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide(I-24)

Compound I-24 was prepared in a manner similar to the synthesis ofCompound I-23 as shown in Example 26. MS m/z: 1097.92 [M+1]⁺; ¹H NMR(500 MHz, DMSO-d₆) δ=12.70 (br s, 1H), 8.98 (s, 1H), 8.50 (d, J=2.7 Hz,1H), 8.37 (d, J=7.6 Hz, 1H), 7.94-7.90 (m, 2H), 7.88-7.84 (m, 2H), 7.61(d, J=8.2 Hz, 1H), 7.51-7.35 (m, 10H), 7.28 (d, J=3.7 Hz, 1H), 6.92 (d,J=9.2 Hz, 1H), 6.33 (s, 1H), 5.10 (d, J=3.4 Hz, 1H), 4.96-4.88 (m, 1H),4.78 (d, J=17.7 Hz, 1H), 4.53 (d, J=9.5 Hz, 1H), 4.43 (t, J=8.1 Hz, 1H),4.28 (br s, 1H), 4.00 (d, J=17.7 Hz, 1H), 3.67-3.45 (m, 15H), 2.58-2.51(m, 6H), 2.45 (s, 3H), 2.38-2.32 (m, 1H), 2.04-1.98 (m, 1H), 1.79 (ddd,J=4.6, 8.2, 12.8 Hz, 1H), 1.37 (d, J=7.0 Hz, 3H), 0.94 (s, 9H).

Example 28: Alternative Synthesis of(2S,4R)-1-((2S)-3,3-Dimethyl-2-(2-(4-(4-(3-oxo-2-(2-oxo-1-phenyl-2-(thiazol-2-ylamino)ethyl)isoindolin-5-yl)phenyl)piperazin-1-yl)acetamido)butanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide(I-6)

Compound I-6 was prepared in a manner similar to the synthesis ofCompound I-23 as shown in Example 26. MS m/z: 980.55 [M+1]⁺.

Example 29: Synthesis of(2S,4R)-1-((2S)-2-(tert-Butyl)-4,16-dioxo-16-(4-(4-(3-oxo-2-(2-oxo-1-phenyl-2-(thiazol-2-ylamino)ethyl)isoindolin-5-yl)phenyl)piperazin-1-yl)-7,10,13-trioxa-3-azahexadecanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide(I-25)

Step 1:(S)-15-((2S,4R)-4-Hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidine-1-carbonyl)-16,16-dimethyl-13-oxo-4,7,10-trioxa-14-azaheptadecanoicacid (2-77)

2,2-dimethyl-4-oxo-3,7,10,13-tetraoxahexadecan-16-oic acid (107 mg, 0.35mmol), 2-71 (100 mg, 0.23 mmol) and HATU (175 mg, 0.46 mmol) weredissolved in DMF (2.5 mL), and DIEA (160 μL, 0.92 mmol) was added to themixture. After stirring for 30 minutes, the reaction mixture was dilutedwith EtOAc and washed with water five times. The organic layer was driedover Na₂SO₄, filtered and concentrated. The residue was purified bypreparative HPLC to afford tert-butyl(S)-15-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidine-1-carbonyl)-16,16-dimethyl-13-oxo-4,7,10-trioxa-14-azaheptadecanoateas an off-white solid.

To a solution of tert-butyl(S)-15-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidine-1-carbonyl)-16,16-dimethyl-13-oxo-4,7,10-trioxa-14-azaheptadecanoatein DCM (7 mL) was added TFA (3 mL). After stirring for 1.5 hours, thereaction mixture was concentrated and purified by flash columnchromatography (DCM:1.75 N NH₃ in MeOH=100:0 to 80:20) to afford 2-77(108 mg, 70%) as a white solid.

Step 2:(2S,4R)-1-((2S)-2-(tert-Butyl)-4,16-dioxo-16-(4-(4-(3-oxo-2-(2-oxo-1-phenyl-2-(thiazol-2-ylamino)ethyl)isoindolin-5-yl)phenyl)piperazin-1-yl)-7,10,13-trioxa-3-azahexadecanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide(1-25)

To a solution of TL compound 1 (61 mg, 0.12 mmol) and 2-77 (53 mg, 0.08mmol) in DMF (1 mL) was added HATU (61 mg, 0.16 mmol) and DIEA (56 μL,0.32 mmol). After stirring overnight, the reaction mixture was purifiedby preparative HPLC to afford I-25 (50 mg, 52%) as a white solid. MSm/z: 1154.84 [M+1]⁺; ¹H NMR (500 MHz, DMSO-d₆) δ=12.70 (br s, 1H), 8.98(s, 1H), 8.56 (t, J=6.3 Hz, 1H), 7.95-7.81 (m, 3H), 7.65-7.55 (m, 3H),7.54-7.31 (m, 10H), 7.28 (d, J=3.4 Hz, 1H), 7.05 (d, J=8.5 Hz, 2H), 6.33(s, 1H), 5.12 (d, J=3.7 Hz, 1H), 4.77 (d, J=17.7 Hz, 1H), 4.55 (d, J=9.5Hz, 1H), 4.46-4.39 (m, 2H), 4.37-4.32 (m, 1H), 4.21 (dd, J=5.5, 15.6 Hz,1H), 3.99 (d, J=17.7 Hz, 1H), 3.69-3.53 (m, 10H), 3.51-3.43 (m, 8H),3.25-3.20 (m, 2H), 3.19-3.14 (m, 2H), 2.62 (t, J=6.6 Hz, 2H), 2.57-2.53(m, 1H), 2.44 (s, 3H), 2.39-2.32 (m, 1H), 2.07-2.00 (m, 1H), 1.90 (ddd,J=4.3, 8.5, 12.9 Hz, 1H), 0.92 (s, 9H).

Example 30: Synthesis of(2S,4R)-1-((2S)-2-(tert-Butyl)-16-(4-(4-(2-(1-(5-fluoro-2-hydroxyphenyl)-2-oxo-2-(thiazol-2-ylamino)ethyl)-3-oxoisoindolin-5-yl)phenyl)piperazin-1-yl)-4,16-dioxo-7,10,13-trioxa-3-azahexadecanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide(I-26)

Compound I-26 was prepared in a manner similar to the synthesis ofCompound I-25 as shown in Example 29. Step 2 of the synthesis ofCompound I-26 is shown above in Example 30. MS m/z: 1189.12 [M+1]⁺; ¹HNMR (500 MHz, DMSO-d₆) δ=9.96 (s, 1H), 8.98 (s, 1H), 8.56 (t, J=5.6 Hz,1H), 7.93-7.83 (m, 3H), 7.65-7.57 (m, 3H), 7.49 (d, J=3.4 Hz, 1H),7.44-7.35 (m, 4H), 7.27 (d, J=3.1 Hz, 1H), 7.14-7.06 (m, 1H), 7.05 (d,J=8.5 Hz, 2H), 6.91 (dd, J=8.7, 4.7 Hz, 1H), 6.86 (dd, J=9.0, 2.6 Hz,1H), 6.33 (s, 1H), 5.12 (br s, 1H), 4.63 (d, J=17.1 Hz, 1H), 4.55 (d,J=9.5 Hz, 1H), 4.47-4.39 (m, 2H), 4.35 (br s, 1H), 4.21 (dd, J=15.7, 5.3Hz, 1H), 3.99 (d, J=17.4 Hz, 1H), 3.73-3.53 (m, 10H), 3.53-3.40 (m, 8H),3.23 (br s, 2H), 3.17 (br s, 2H), 2.62 (t, J=6.6 Hz, 2H), 2.57-2.53 (m,1H), 2.44 (s, 3H), 2.39-2.29 (m, 1H), 2.08-1.98 (m, 1H), 1.96-1.83 (m,1H), 0.93 (s, 9H).

Example 31. Synthesis of4-Fluoro-2-(3-methyl-2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione(2-81)

Step 1: tert-Butyl (3-methyl-2,6-dioxopiperidin-3-yl)carbamate (2-79)

To a solution of tert-butyl (2-78, 2,6-dioxopiperidin-3-yl)carbamate(1.0 g, 4.38 mmol) in THE (15 mL) was added NaH (263 mg, 6.57 mmol) at0° C. After stirring for 20 minutes at room temperature, iodomethane(327 μL, 5.26 mmol) was added to the reaction mixture. After stirringfor 2 hours, the mixture was diluted with EtOAc and quenched with water.The organic layer was collected and the aqueous layer was washed withEtOAc. The combined organic layers were dried over Na₂SO₄, filtered andconcentrated. The crude compound 2-79 was carried forward in the nextstep without additional purification.

Step 2: 3-Amino-3-methylpiperidine-2,6-dione HCl salt (2-80)

Compound 2-79 was dissolved in dioxane and 4M HCl in dioxane was addedto the resulting solution. After stirring for 2 hours, the reactionmixture was concentrated under reduced pressure to afford crude productwhich was carried forward in the next step without additionalpurification.

Step 3:4-Fluoro-2-(3-methyl-2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione(2-81)

Compound 2-80 and 4-fluoroisobenzofuran-1,3-dione were dissolved inacetic acid. The resulting mixture was stirred at 100° C. for overnightand the reaction mixture was concentrated under reduced pressure. Theresidue was re-dissolved in DCM and basified with saturated NaHCO₃. Theaqueous layer was washed with DCM three times. Combined organic layerwas dried over Na₂SO₄, filtered and concentrated under reduced pressure.The residue was purified by flash column chromatography to affordcompound 2-81 (432 mg, 34%, three steps) as a white solid. ¹H NMR (500MHz, DMSO-d₆) δ=10.97 (s, 1H), 7.88-7.81 (m, 1H), 7.69-7.59 (m, 1H),2.69-2.45 (m, 3H), 2.04-1.95 (m, 1H).

Example 32. Synthesis of2-(6-(4-(4-(3-(2-(2-(2-((2-(3-Methyl-2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)ethoxy)propanoyl)piperazin-1-yl)phenyl)-1-oxoisoindolin-2-yl)-2-phenyl-N-(thiazol-2-yl)acetamide(1-27)

Compound I-27 was prepared according to the synthesis of Compound I-1 asshown in Example 3. MS m/z: 983.48 [M+1]⁺; ¹H NMR (500 MHz, DMSO-d₆)δ=12.70 (s, 1H), 10.97 (s, 1H), 7.91-7.82 (m, 2H), 7.67-7.57 (m, 3H),7.55-7.41 (m, 5H), 7.39 (d, J=7.3 Hz, 2H), 7.28 (d, J=3.1 Hz, 1H), 7.07(d, J=8.5 Hz, 1H), 7.04 (d, J=8.5 Hz, 2H), 6.94 (d, J=7.0 Hz, 1H), 6.60(t, J=5.5 Hz, 1H), 6.33 (s, 1H), 4.77 (d, J=17.4 Hz, 1H), 3.99 (d,J=17.4 Hz, 1H), 3.69-3.55 (m, 8H), 3.55-3.47 (m, 6H), 3.44-3.39 (m, 2H),3.22 (br s, 2H), 3.16 (br s, 2H), 2.74-2.45 (m, 7H), 2.06-1.95 (m, 1H),1.87 (s, 3H).

Example 33. Synthesis of2-(6-(4-(4-(3-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)propanoyl)piperazin-1-yl)phenyl)-1-oxoisoindolin-2-yl)-2-(5-fluoro-2-hydroxyphenyl)-N-(thiazol-2-yl)acetamide(I-28)

Compound I-28 was prepared in a manner similar to the synthesis ofCompound I-3 as shown in Example 9. Step 4 of the synthesis of CompoundI-28 is shown above in Example 33. MS m/z: 959.43 [M+1]⁺; ¹H NMR 500 MHz(DMSO-d₆) δ 11.10 (s, 1H), 7.88-7.81 (m, 2H), 7.63-7.54 (m, 4H), 7.38(d, J=3.1 Hz, 1H), 7.13 (d, J=8.5 Hz, 1H), 7.10-7.01 (m, 4H), 6.89-6.82(m, 2H), 6.60 (br t, J=5.3 Hz, 1H), 6.20 (s, 1H), 5.05 (dd, J=12.6, 5.2Hz, 1H), 4.80 (d, J=17.7 Hz, 1H), 4.22 (d, J=17.7 Hz, 1H), 3.68-3.40 (m,10H), 3.22-3.12 (m, 4H), 2.92-2.82 (m, 1H), 2.65-2.50 (m, 4H), 2.06-1.97(m, 1H).

Example 34. Synthesis of2-(6-(4-(4-(3-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)propanoyl)piperazin-1-yl)phenyl)-1-oxoisoindolin-2-yl)-2-(5-fluoro-2-hydroxyphenyl)-N-(thiazol-2-yl)acetamide(I-29)

Compound I-29 was prepared according to the synthesis of Compound I-3 asshown in Example 9. Step 4 of the synthesis of Compound I-29 is shownabove in Example 34. MS m/z: 915.45 [M+1]⁺; ¹H NMR 500 MHz (DMSO-d₆) δ11.10 (bs, 1H), 7.83 (bs, 2H) 7.64 (d, J=8.9 Hz, 1H), 7.69-7.53 (m, 3H),7.23 (d, J=3.4 Hz, 1H), 7.13 (d, J=8.9 Hz, 1H), 7.05-6.97 (m, 3H),6.94-6.88 (m, 1H), 6.85-6.80 (m, 1H), 6.79-6.72 (m, 2H), 6.58 (br t,J=5.0 Hz, 1H), 6.02 (s, 1H), 5.12-5.00 (m, 2H), 4.54 (d, J=18.3 Hz, 1H),3.71 (t, J=6.6 Hz, 2H), 3.65-3.57 (m, 6H), 3.49-3.43 (m, 4H), 2.92-2.82(m, 1H), 2.64 (t, J=6.3 Hz, 2H), 2.60-2.50 (m, 2H), 2.05-1.96 (m, 1H).

Example 35. Synthesis of2-(6-(4-(4-(1-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)-3,6,9,12-tetraoxapentadecan-15-oyl)piperazin-1-yl)phenyl)-1-oxoisoindolin-2-yl)-2-(5-fluoro-2-hydroxyphenyl)-N-(thiazol-2-yl)acetamide(I-30)

Compound I-30 was prepared according to the synthesis of Compound I-3 asshown in Example 9. Step 4 of the synthesis of Compound I-30 is shownabove in Example 35. MS m/z: 1047.45 [M+1]⁺; ¹H NMR 500 MHz (DMSO-d₆) δ12.61 (bs, 1H), 11.09 (s, 1H), 10.02 (bs, 1H), 7.88-7.87 (m, 1H), 7.86(dd, J=7.9, 1.5 Hz, 1H), 7.65 (d, J=8.9 Hz, 2H), 7.62-7.54 (m, 2H), 7.49(d, J=3.7 Hz, 1H), 7.27 (d, J=3.7 Hz, 1H), 7.17-7.08 (m, 4H), 7.03 (d,J=7.0 Hz, 1H), 6.94 (dd, J=9.2, 4.9 Hz, 1H), 6.86 (dd, J=9.2, 3.7 Hz,1H), 6.59 (br, 1H), 6.33 (s, 1H), 5.05 (dd, J=12.8, 5.5 Hz, 1H), 4.63(d, J=17.4 Hz, 1H), 4.00 (d, J=17.4 Hz, 1H), 3.68-3.42 (m, 22H),3.28-3.17 (m, 4H), 2.92-2.83 (m, 1H), 2.65-2.50 (m, 4H), 2.34-2.27 (m,2H), 2.05-1.98 (m, 1H).

Example 36. Synthesis of2-(6-(4-(4-(2-(2-(2-(2-((2-(3-methyl-2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)ethoxy)ethyl)piperazin-1-yl)phenyl)-1-oxoisoindolin-2-yl)-2-phenyl-N-(thiazol-2-yl)acetamide(I-31)

Compound I-31 was prepared according to the synthesis of Compound I-18as shown in Example 21. Step 3 of the synthesis of Compound I-31 isshown above in Example 36. MS m/z: 955.59 [M+1]⁺.

Example 37. Synthesis of2-(6-(6-(4-(2-(2-(2-(2-((2-(3-methyl-2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)ethoxy)ethyl)piperazin-1-yl)pyridin-3-yl)-1-oxoisoindolin-2-yl)-2-phenyl-N-(thiazol-2-yl)acetamide(I-32)

Compound I-32 was prepared according to the synthesis of Compound I-22as shown in Example 25. Step 3 of the synthesis of Compound I-32 isshown above in Example 37. MS m/z: 956.61 [M+1]⁺; ¹H NMR (500 MHz,DMSO-d₆) δ=12.70 (s, 1H), 10.98 (s, 1H), 8.49 (d, J=2.1 Hz, 1H),7.97-7.88 (m, 2H), 7.86 (dd, J=7.9, 1.5 Hz, 1H), 7.61 (d, J=8.2 Hz, 1H),7.54 (dd, J=8.4, 7.2 Hz, 1H), 7.51-7.41 (m, 4H), 7.41-7.36 (m, 2H), 7.28(d, J=3.4 Hz, 1H), 7.10 (d, J=8.5 Hz, 1H), 6.95 (d, J=7.2 Hz, 1H),6.93-6.88 (m, 1H), 6.62 (t, J=5.8 Hz, 1H), 6.32 (s, 1H), 4.78 (d, J=17.7Hz, 1H), 4.00 (d, J=17.7 Hz, 1H), 3.62 (t, J=5.5 Hz, 2H), 3.60-3.47 (m,16H), 3.47-3.40 (m, 2H), 2.75-2.43 (m, 7H), 2.04-1.96 (m, 1H), 1.87 (s,3H).

Example 38. Synthesis of(2S,4R)-1-((2S)-2-(tert-Butyl)-4,16-dioxo-16-(4-(4-(3-oxo-2-(2-oxo-1-phenyl-2-(thiazol-2-ylamino)ethyl)isoindolin-5-yl)phenyl)piperazin-1-yl)-7,10,13-trioxa-3-azahexadecanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide(I-34)

To a solution of TL compound 1 (61 mg, 0.12 mmol) and 2-77 (53 mg, 0.08mmol) in DMF (1 mL) were added HATU (61 mg, 0.16 mmol) and DIEA (56 μL,0.32 mmol). After stirring overnight, the reaction mixture was purifiedby preparative HPLC to afford I-34 (50 mg, 52%) as a white solid. MSm/z: 1154.84 [M+1]⁺; ¹H NMR (500 MHz, DMSO-d₆) δ=12.70 (br s, 1H), 8.98(s, 1H), 8.56 (t, J=6.3 Hz, 1H), 7.95-7.81 (m, 3H), 7.65-7.55 (m, 3H),7.54-7.31 (m, 10H), 7.28 (d, J=3.4 Hz, 1H), 7.05 (d, J=8.5 Hz, 2H), 6.33(s, 1H), 5.12 (d, J=3.7 Hz, 1H), 4.77 (d, J=17.7 Hz, 1H), 4.55 (d, J=9.5Hz, 1H), 4.46-4.39 (m, 2H), 4.37-4.32 (m, 1H), 4.21 (dd, J=5.5, 15.6 Hz,1H), 3.99 (d, J=17.7 Hz, 1H), 3.69-3.53 (m, 10H), 3.51-3.43 (m, 8H),3.25-3.20 (m, 2H), 3.19-3.14 (m, 2H), 2.62 (t, J=6.6 Hz, 2H), 2.57-2.53(m, 1H), 2.44 (s, 3H), 2.39-2.32 (m, 1H), 2.07-2.00 (m, 1H), 1.90 (ddd,J=4.3, 8.5, 12.9 Hz, 1H), 0.92 (s, 9H).

Example 39. EGFR Protein Expression and Purification

Constructs spanning residues 696-1022 of the human EGFR (including wildtype and L858R, L858R/T790M, T790M, and T790M/V948R mutant sequences)were prepared in a GST-fusion format using the pTriEX system (Novagen)for expression in Sf9 insect cells essentially as described. (Yun, C. H.et al. The T790M mutation in EGFR kinase causes drug resistance byincreasing the affinity for ATP. Proc Natl Acad Sci USA 105, 2070-2075(2008); Yun, C. H. et al. Structures of lung cancer-derived EGFR mutantsand inhibitor complexes: mechanism of activation and insights intodifferential inhibitor sensitivity. Cancer Cell 11, 217-227 (2007)) EGFRkinase proteins were purified by glutathione-affinity chromatographyfollowed by size-exclusion chromatography after cleavage with TEV orthrombin to remove the GST fusion partner following establishedprocedures. (Yun, C. H. et al. Proc Natl Acad Sci USA 105, 2070-2075(2008); Yun, C. H. et al. Cancer Cell 11, 217-227 (2007)

Example 40. H1975, H3255 & HaCaT Target Modulation Assays Tissue Culture

Cells were maintained in 10% FBS/RPMI supplemented with 100 μg/mLPenicillin/Streptomycin (Hyclone #SH30236.01). The cells were harvestedwith 0.25% Trypsin/EDTA (Hyclone #SH30042.1), re-suspended in 5%FBS/RPMI Pen/Strep and plated at 7,500 cells per well in 50 μL of mediain a 384-well black plate with clear bottoms (Greiner #789068G). Thecells were allowed to incubate overnight in a 37° C., 5% CO₂ humidifiedtissue culture incubator. The 12-point serial diluted test compoundswere transferred to the plate containing cells by using a 50 nL Pin Headdevice (Perkin Elmer) and the cells were placed back in the incubatorfor 3 hours.

Phospho-EGFR (Y1173) Target Modulation Assay

HaCaT cells were stimulated with 10 ng/mL EGF (Peprotech # AF-100-15)for 5 minutes at room temperature. Constitutively activated EGFR mutantcell lines (H1975 and H3255) were not stimulated with EGF. The media wasreduced to 20 μL using a Bio-Tek ELx 405 Select™ plate washer. Cellswere lysed with 20 μL of 2× Lysis buffer containing protease andphosphatase inhibitors (2% Triton X-100, 40 mM Tris, pH 7.5, 2 mM EDTA,2 mM EGTA, 300 mM NaCl, 2× complete cocktail inhibitor (Roche #11 697498 001), 2× Phosphatase Inhibitor Cocktail Set II and Set III (Sigma#P5726 and #P0044)). The plates were shaken for 20 minutes. An aliquotof 25 μL from each well was transferred to prepared ELISA plates foranalysis.

For the experiment studying the effect of EGF pre-treatment on compound(e.g., compounds of the present application) target modulation, H1975cells were harvested and plated in 0.5% FBS/RPMI Pen/Strep. On thefollowing day, cells were pre-treated with 0.5% FBS/RPMI media with orwithout 10 ng EGF/mL for 5 minutes. Compound (i.e., compounds of thepresent application) was added and assay was carried out as describedabove.

Phospho-EGFR (Y1173) ELISA

Solid white 384-well high-binding ELISA plates (Greiner #781074) werecoated with 5 μg/mL goat anti-EGFR capture antibody overnight in 50 mMcarbonate/bicarbonate pH 9.5 buffer. Plates were blocked with 1% BSA(Sigma #A7030) in PBS for 1 hour at room temperature, and washes werecarried out with a Bio-Tek ELx405 Select™ using 4 cycles of 100 μL TBS-T(20 mM Tris, 137 mM NaCl, 0.05% Tween-20) per well. A 25 μL aliquot oflysed cell was added to each well of the ELISA plate and incubatedovernight at 4° C. with gentle shaking. A 1:1,000 anti-phospho-EGFR in0.2% BSA/TBS-T was added and incubated for 2 hours at room temperature.After washing, 1:2,000 anti-rabbit-HRP in 0.2% BSA/TBS-T was added andincubated for 1 hour at room temperature. Chemiluminescent detection wascarried out with SuperSignal ELISA Pico substrate. Signal was read onEnVision plate reader using built-in UltraLUM setting.

Example 41. Western Blotting

Cell lysates were equalized to protein content determined by CoomassiePlus' Protein Assay Reagent (ThermoScientific #1856210) and loaded onto4-12% NuPAGE Bis-Tris gels with MOPS running buffer with LDS Samplebuffer (supplemented with DTT. Gel proteins were transferred to PVDFmembranes with an iBlot® Gel Transfer Device. 1× Casein-blockedmembranes were probed with primary antibodies overnight at 4° C. on anend-over-end rotisserie. Membranes were washed with TBS-T andHRP-conjugated secondary antibodies were added for 1 hour at roomtemperature. After washing, HRP was detected using Luminata™ ForteWestern HRP Substrate reagent and recorded with a Bio-Rad VersaDocimager.

EGFR protein degradation was assessed by western blotting aftertreatment of T790M/L858R mutant Ba/F3 cell lines with a compound of thepresent application dose-dependently for 8 hour or in combination with 1ug/mL of cetuximab. The results are shown in FIG. 3.

Example 42. Proliferation Assay

H1975, H3255 and HaCaT cell lines were plated in solid white 384-wellplates (Greiner) at 500 cells per well in 10% FBS RPMI P/S media. Usinga Pin Tool, 50 nL of serial diluted compounds of the present applicationwere transferred to the cells. After 3 days, cell viability was measuredby CellTiter-Glo (Promega) according to manufacturer's instructions.Luminescent readout was normalized to 0.1% DMSO-treated cells and emptywells. Data was analyzed by non-linear regression curve-fitting and EC₅₀values were reported. Inhibition data of an EGFR T790M/L858R cell linefor exemplary compounds of the present application combined withcetuximab (% cetuximab, 1.0 μM drug concentration with 1.0 μg/mLcetuximab) can be found in Table 2 below.

Ba/F3 Cell Proliferation Models

The EGFR mutant L858R, Del E746_A750, L858R/T790M, DelE746_A750/T790M,L858R/T790M/C797S and Del/T790M/C797S Ba/F3 cells have been previouslydescribed (Zhou, W., Ercan, D., Chen, L., Yun, C. H., Li, D.,Capelletti, M., Cortot, A. B., Chirieac, L., Iacob, R. E., Padera, R.,et al. “Novel mutant-selective EGFR kinase inhibitors against EGFRT790M,” Nature 462, (2009), 1070-1074). All cell lines were maintainedin RPMI 1640 (Cellgro; Mediatech Inc., Herndon, Calif.) supplementedwith 10% FBS 100 units/mL penicillin, 100 units/mL streptomycin, and 2mM glutamine. L858R cells were maintained in ACL-4 media (Invitrogen,Carlsbad, Calif.) supplemented with 5% FBS, 100 units/mL penicillin, 100units/mL streptomycin, and 2 mM glutamine. The EGFR I941R mutation wasintroduced via site directed mutagenesis using the Quick ChangeSite-Directed Mutagenesis kit (Stratagene; La Jolla, Calif.) accordingto the manufacturer's instructions. All constructs were confirmed by DNAsequencing. The constructs were shuttled into the retroviral vectorJP1540 using the BD Creator System (BD Biosciences). Ba/F3 cells wereinfected with retrovirus and according to standard protocols, asdescribed previously (Zhou et al, Nature 2009). Stable clones wereobtained by selection in puromycin (2 g/ml).

Growth and inhibition of growth was assessed by MTS assay and wasperformed according to previously established methods (Zhou et al.,Nature 2009). The MTS assay is a colorimetric method for determining thenumber of viable cells that is based on the bioreduction of MTS by cellsto a formazan product that is soluble in cell culture medium and can bedetected spectrophotometrically. Ba/F3 cells of different EGFR genotypeswere exposed to treatment for 72 hours and the number of cells used perexperiment determined empirically and has been previously established(Zhou et al., Nature 2009). All experimental points were set up in sixwells and all experiments were repeated at least three times. The datawas graphically displayed using GraphPad Prism version 5.0 for Windows,(GraphPad Software; www.graphpad.com). The curves were fitted using anon-linear regression model with a sigmoidal dose response.

Example 43. Mouse Efficacy Studies

EGFR-TL (T790M/L858R) and EGFR-TD (exon 19 deletion-T790M) mice weregenerated as previously described (Li, D., Shimamura, T., Ji, H., Chen,L., Haringsma, H. J., McNamara, K., Liang, M. C., Perera, S. A.,Zaghlul, S., Borgman, C. L., et al., “Bronchial and peripheral murinelung carcinomas induced by T790M-L858R mutant EGFR respond to HKI-272and rapamycin combination therapy,” Cancer Cell 12, (2007), 81-93; Zhouet al., Nature 2009). The EGFR-L858R;T790M;C797S (“TLCS”) mutant mousecohort was established briefly as follows: The full-length HuTLCS cDNAwas generated by site-directed mutagenesis using the Quickchange sitedirected mutagenesis kit (Agilent Technologies) and further verified byDNA sequencing. Sequence-verified targeting vectors wereco-electroporated with an FLPe recombinase plasmid into v6.5 C57BL/6J(female)×129/sv (male) embryonic stem cells (Open Biosystems) asdescribed elsewhere (Beard, C., Hochedlinger, K., Plath, K., Wutz, A.,and Jaenisch, R., “Efficient method to generate single-copy transgenicmice by site-specific integration in embryonic stem cells,” Genesis 44,(2006), 23-28). Resulting hygromycin-resistant embryonic stem cloneswere evaluated for transgene integration via PCR. Then,transgene-positive embryonic stem clones were injected into C57BL/6blastocysts, and the resulting chimeras were mated with BALB/c WT miceto determine germline transmission of the TLCS transgene. Progeny of TL,TD and TLCS mice were genotyped by PCR of tail DNA.

The TL and TD mice were fed a doxycycline diet at 6 weeks of age toinduce EGFR TL or TD expression, respectively. The TLCS mice wereintranasally instilled with Ad-Cre (University of Iowa viral vectorcore) at 6 weeks of age to excise the loxP sites, activating EGFR TLCSexpression.

All care of experimental animals was in accordance with Harvard MedicalSchool/Dana-Farber Cancer Institute (DFCI) institutional animal care anduse committee (IACUC) guidelines. All mice were housed in apathogen-free environment at a DFCI animal facility and handled instrict accordance with Good Animal Practice as defined by the Office ofLaboratory Animal Welfare.

Example 44. In Vivo Treatment and MRI Tumor Volume Quantification

The TL, TD and TLCS mice were monitored by MRI to quantify lung tumorburden before being assigned to various treatment study cohorts. All thetreatment mice had equal amount initial tumor burden. A compound of thepresent application was dissolved in 10% NMP (10%1-methyl-2-pyrrolidinone: 90% PEG-300), and was dosed at 60 mg/kg dailyby oral gavage. Cetuximab was administrated at 1 mg/mouse every threedays by intraperitoneal in injection. MRI evaluation was repeated every2 weeks during the treatment. The animals were imaged with a rapidacquisition with relaxation enhancement sequence (TR=2000 ms, TEeffect=25 ms) in the coronal and axial planes with a 1-mm slicethickness gating with respiratory rates. The detailed procedure for MRIscanning has been previously described (Li et al., 2007). The tumorburden volumes were quantified using 3-dimensional Slicer software.

Example 45. Bifunctional Compounds are Efficacious Against T790M/L858RTransformed Ba/F3 Cells

Compounds of Table 1 were tested at a concentration of 1 μM againstT790M/L858R transformed Ba/F3 cells. The assay, as described in Example42, measured inhibition of growth as a percent of the inhibitoryactivity of the control. In Table 1, A is 0<% control <25, B is 25<%control <50, C is 50<% control <75, and D is 75<% control.

TABLE 1 Antiproliferative activity against T790M/L858R transformed Ba/F3cells Compound % control I-28 A I-26 D I-10 B I-11 B I-12 B I-33 A I-29A I-27 D I-25 D I-7 B I-17 C I-18 A I-19 D I-20 A I-35 D I-8 D I-21 CI-1 A I-23 D I-22 A I-15 C I-16 C I-14 D TL Compd 1 B TL Compd 2 D

Example 46. Bifunctional Compounds are Potent Against T790M/L858RTransformed Ba/F3 Cells

Compounds of Table 2 were tested in a T790M/L858R transformed Ba/F3 cellline as described in Example 42 and the EC₅₀ values were calculated. InTable 2, A is EC₅₀<500 nM, B is 500 nM<EC₅₀<1000 nM, C is 1000nM<EC₅₀<5000 nM, and D is 5000 nM<EC₅₀.

TABLE 2 Antiproliferative activity against T790M/L858R transformed Ba/F3cells Compound T790M/L858R (EC₅₀) I-29 B I-28 B I-33 A I-1 A I-10 C I-18A I-20 A I-11 C I-12 C I-15 C I-16 C I-22 A I-14 C I-23 D I-24 D

Example 47. Bifunctional Compounds are Potent Against T790M/L858R/C797STransformed Ba/F3 Cells

Compounds of Table 3 were tested in a T790M/L858R transformed Ba/F3 cellline as described in Example 42 and the EC₅₀ values were calculated. InTable 3, A is EC₅₀<500 nM, B is 500 nM<EC₅₀<1000 nM, C is 1000nM<EC₅₀<5000 nM, and D is 5000 nM<EC₅₀.

TABLE 3 Antiproliferative activity against T790M/L858R/C797S transformedBa/F3 cells Compound T790M/L858R/C797S (EC₅₀) I-29 D I-28 D I-33 C I-1 BI-10 D I-18 A I-20 A I-11 D I-12 D I-15 D I-16 D I-22 B I-14 D I-23 DI-24 D

This specification has been described with reference to embodiments ofthe invention. However, one of ordinary skill in the art appreciatesthat various modifications and changes can be made without departingfrom the scope of the invention as set forth in the claims below.Accordingly, the specification was to be regarded in an illustrativerather than a restrictive sense, and all such modifications are intendedto be included within the scope of invention.

1. A bifunctional compound of the Formula I:

or an enantiomer, diastereomer, stereoisomer, or pharmaceuticallyacceptable salt thereof, wherein: the Linker is of Formula L0:

wherein the Linker is covalently bonded to the Degron via the

 next to Q, and covalently bonded to

 via the

 next to Z; the Degron is Formula D1c:

A is phenyl or pyridinyl; X is CH, C(C₁-C₃) alkyl, or N; R¹ is H or(C₁-C₃) alkyl; R² is (C₆-C₁₀) aryl, or heteroaryl comprising one or two5- to 7-membered rings and 1-4 heteroatoms selected from N, O, and S,wherein the aryl and heteroaryl are each optionally substituted with oneor more R⁴; each R⁴ is independently selected from (C₁-C₄) alkyl,(C₁-C₄) haloalkyl, (C₁-C₄) alkoxy, (C₁-C₄) haloalkoxy, halogen, NO₂, OH,CN, C(O)R⁶, C(O)OR⁶, C(O)NR⁶R⁷, NR⁶R⁷, (C₃-C₇) cycloalkyl, heterocyclylcomprising a 5- to 7-membered ring and 1-3 heteroatoms selected from N,O, and S, (C₆-C₁₀) aryl, and heteroaryl comprising one or two 5- to7-membered rings and 1-4 heteroatoms selected from N, O, and S, whereinthe alkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl are eachoptionally substituted with one or more R⁵; each R⁵ is independentlyselected from (C₁-C₄) alkyl, (C₁-C₄) haloalkyl, (C₁-C₄) alkoxy, (C₁-C₄)haloalkoxy, halogen, NO₂, OH, and CN; each R⁶ is independently H or(C₁-C₃) alkyl; each R⁷ is independently H or (C₁-C₃) alkyl; R³ is(C₁-C₃) alkyl or

X² is N or CR⁸; R⁸ is H, (C₁-C₄) alkyl, (C₁-C₄) haloalkyl, (C₁-C₄)alkoxy, (C₁-C₄) haloalkoxy, halogen, NO₂, NH₂, OH, or CN; each R₉ isindependently selected from (C₁-C₄) alkyl, (C₁-C₄) haloalkyl, (C₁-C₄)alkoxy, (C₁-C₄) haloalkoxy, halogen, NO₂, NH₂, OH, and CN; n is 0 or 1;p is 0, 1, 2, or 3; p1 is an integer selected from 0 to 12; p2 is aninteger selected from 0 to 12; p3 is an integer selected from 0 to 6;each W is independently absent, CH₂, or O; Z is absent, CH₂, O, orC(O)(CH₂)₀₋₃; Q is absent or heterocycle; each R¹⁴ is independentlyC₁-C₃ alkyl; each R¹⁶ is independently halogen, OH, C₁-C₆ alkyl, orC₁-C₆ alkoxy; q is 0, 1, or 2; and v is 0, 1, 2, or
 3. 2. (canceled) 3.The bifunctional compound of claim 1, wherein X is N.
 4. Thebifunctional compound of claim 1, wherein A is phenyl.
 5. Thebifunctional compound of claim 1, wherein n is
 0. 6. The bifunctionalcompound of claim 1, wherein R³ is


7. The bifunctional compound of claim 1, wherein X² is CH.
 8. Thebifunctional compound of claim 1, wherein R¹ is H.
 9. (canceled)
 10. Thebifunctional compound of claim 1, wherein R² is thiazole. 11-14.(canceled)
 15. The bifunctional compound of claim 1, wherein thecompound is of Formula:

or an enantiomer, diastereomer, stereoisomer, or pharmaceuticallyacceptable salt thereof.
 16. The bifunctional compound of claim 1,wherein R² is


17. (canceled)
 18. The bifunctional compound of claim 1, wherein theLinker is selected from:


19. (canceled)
 20. The bifunctional compound of claim 1, wherein theLinker is selected from:

21-30. (canceled)
 31. A pharmaceutical composition comprising atherapeutically effective amount of the bifunctional compound of claim1, or an enantiomer, diastereomer, stereoisomer, or pharmaceuticallyacceptable salt thereof, and a pharmaceutically acceptable carrier.32-36. (canceled)
 37. A method of treating cancer or a proliferationdisease, comprising administering to a subject in need thereof aneffective amount of a compound of claim
 1. 38. (canceled)
 39. The methodof claim 37, wherein the cancer is lung cancer, colon cancer, breastcancer, prostate cancer, liver cancer, pancreas cancer, brain cancer,kidney cancer, ovarian cancer, stomach cancer, skin cancer, bone cancer,gastric cancer, breast cancer, pancreatic cancer, glioma, glioblastoma,hepatocellular carcinoma, papillary renal carcinoma, head and necksquamous cell carcinoma, leukemias, lymphomas, myelomas, or solidtumors.
 40. The method of claim 37, wherein the disease is inflammation,arthritis, rheumatoid arthritis, spondyloarthropathies, gouty arthritis,osteoarthritis, juvenile arthritis, and other arthritic conditions,systemic lupus erythematosus (SLE), skin-related conditions, psoriasis,eczema, burns, dermatitis, neuroinflammation, allergy, pain, neuropathicpain, fever, pulmonary disorders, lung inflammation, adult respiratorydistress syndrome, pulmonary sarcoidosis, asthma, silicosis, chronicpulmonary inflammatory disease, and chronic obstructive pulmonarydisease (COPD), cardiovascular disease, arteriosclerosis, myocardialinfarction (including post-myocardial infarction indications),thrombosis, congestive heart failure, cardiac reperfusion injury, aswell as complications associated with hypertension and/or heart failuresuch as vascular organ damage, restenosis, cardiomyopathy, strokeincluding ischemic and hemorrhagic stroke, reperfusion injury, renalreperfusion injury, ischemia including stroke and brain ischemia, andischemia resulting from cardiac/coronary bypass, neurodegenerativedisorders, liver disease and nephritis, gastrointestinal conditions,inflammatory bowel disease, Crohn's disease, gastritis, irritable bowelsyndrome, ulcerative colitis, ulcerative diseases, gastric ulcers, viraland bacterial infections, sepsis, septic shock, gram negative sepsis,malaria, meningitis, HIV infection, opportunistic infections, cachexiasecondary to infection or malignancy, cachexia secondary to acquiredimmune deficiency syndrome (AIDS), AIDS, ARC (AIDS related complex),pneumonia, herpes virus, myalgias due to infection, influenza,autoimmune disease, graft vs. host reaction and allograft rejections,treatment of bone resorption diseases, osteoporosis, multiple sclerosis,colorectal cancer, epithelial call-derived neoplasia (epithelialcarcinoma), basal cell carcinoma, adenocarcinoma, gastrointestinalcancer, lip cancer, mouth cancer, esophageal cancer, small bowel cancer,bladder cancer, cervical cancer, squamous cell and/or basal cellcancers, renal cell carcinoma, chronic myelogenous leukemia (CML), acutemyeloid leukemia (AML) and acute promyelocytic leukemia (APL),angiogenesis, metastasis, central nervous system disorders, centralnervous system disorders having an inflammatory or apoptotic component,Alzheimer's disease, Parkinson's disease, Huntington's disease,amyotrophic lateral sclerosis, spinal cord injury, and peripheralneuropathy, or B-Cell Lymphoma. 41-42. (canceled)
 43. The bifunctionalcompound of claim 1, wherein the compound is selected from the groupconsisting of

or a pharmaceutically acceptable salt thereof.